25.1 DNA Replication in Chapter 25 DNA Metabolism

25.1 DNA Replication

Long before the structure of DNA was known, scientists wondered at the ability of organisms to create faithful copies of themselves and, later, at the ability of cells to produce many identical copies of large, complex macromolecules. Speculation about these problems centered around the concept of a template, a structure that would allow molecules to be lined up in a specific order and joined to create a macromolecule with a unique sequence and function. The 1940s brought the revelation that DNA was the genetic molecule, but not until James Watson and Francis Crick deduced its structure did the way in which DNA could act as a template for the replication and transmission of genetic information become clear: one strand is the complement of the other. The strict base-pairing rules mean that each strand provides the template for a new strand with a predictable and complementary sequence (see Figs. 8-14, 8-15). The fundamental properties of the DNA replication process and the mechanisms used by the enzymes that catalyze it have proved to be essentially identical in all species.

DNA Replication Follows a Set of Fundamental Rules

Early research on bacterial DNA replication and its enzymes helped to establish several basic properties that have proven applicable to DNA synthesis in every organism.

DNA Replication Is Semiconservative

Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strand. This is semiconservative replication. The semiconservative nature of replication was established by Matthew Meselson and Frank Stahl in 1957.

Replication Begins at an Origin and Usually Proceeds Bidirectionally

Following the confirmation of a semiconservative mechanism of replication, a host of questions arose. Are the parent DNA strands completely unwound before each is replicated? Does replication begin at random places or at a unique point? After initiation at any point in the DNA, does replication proceed in one direction or both?

Photographic images of tritium $(^{3} H)$ $left-parenthesis Superscript 3 Baseline zero width space upper H right-parenthesis$ –labeled bacterial DNA made by John Cairns revealed that the intact chromosome of E. coli is a single huge circle, 1.7 mm long. Radioactive DNA isolated from cells during replication showed an extra loop (Fig. 25-1). Cairns concluded that the loop resulted from the formation of two radioactive daughter strands, each complementary to a parent strand. One or both ends of the loop are dynamic points, termed replication forks, where parent DNA is being unwound and the separated strands quickly replicated. Cairns’s results demonstrated that both DNA strands are replicated simultaneously, and variations on his experiment showed that replication of bacterial chromosomes is bidirectional: both ends of the loop have active replication forks.

A figure shows D N A replication by a circular D N A molecule as an illustration and using electron micrographs. — FIGURE 25-1 Visualization of DNA replication. Stages in the replication of circular DNA molecules have been visualized by electron microscopy. Replication of a circular chromosome produces a structure resembling the Greek letter theta, $θ$ $bold-italic theta$ , as both strands are replicated simultaneously (new strands shown in light red). The electron micrographs show images of plasmid DNA being replicated from a single replication origin. [Electron micrographs: Cairns, J. (1963), The Chromosome of *Escherichia coli, Cold Spring Harbor Symp Quant Biol.,* 28, 43–46. © Cold Spring Harbor Laboratory Press.]

FIGURE 25-1 Visualization of DNA replication. Stages in the replication of circular DNA molecules have been visualized by electron microscopy. Replication of a circular chromosome produces a structure resembling the Greek letter theta, $θ$ $bold-italic theta$ , as both strands are replicated simultaneously (new strands shown in light red). The electron micrographs show images of plasmid DNA being replicated from a single replication origin. [Electron micrographs: Cairns, J. (1963), The Chromosome of *Escherichia coli, Cold Spring Harbor Symp Quant Biol.,* 28, 43–46. © Cold Spring Harbor Laboratory Press.]

A parental duplex consists of two concentric blue circles. An arrow points down to show that a bubble has appeared on the right side. In this bubble, the inner blue strand has moved left on the right side, forming a curved left side the bubble. A short red strand lies against the outer blue strand in place of the piece of blue strand that has curved inward and the inner blue strand ha a similar short red strand against it. The top and bottom edges of the bubble, where the blue strands separate, are labeled replication forks. Lines point from these to a micrograph to the right that shows a circular strand of D N A with a bubble at the upper left. The edges of the bubble, where it joins to the lines of the main circle to the left and right, are labeled replication forks. An arrow points down from the illustration to show a blue circle with a blue internal circle on the left and a red partial circle on the right. The blue circle is neatly curved on the left and has a jagged right side running vertically across the middle of the structure. There is a neat red line curved against the right-hand blue line. A second red line, which is not connected to the right-hand red line, runs against the irregular blue vertical piece following the same jagged pattern. Lines extend from the top and bottom of this chromosome to a micrograph to the right that show a circle with a wavy vertical line running across its enter. An arrow points down to an illustration that has blue circles to the left and right. The right side of the left-hand circle is flattened and the left side of the right-hand circle is flattened, producing flattened sides where the two halves meet. Red curved lines follow the insides of the curved parts of the blue circles but not the flattened sides of the circles, meaning that each red line forms a partial circle missing one side. Lines connect this illustration to a micrograph that shows two circles of D N A joined by a small, curved line that connects then in the center. An arrow points down to show two blue circles that are completely round and joined by a small region where they meet in the center. The internal red lines form almost full circles with small gaps right where the two blue circles join. Two arrows point down, one from the left-hand circle and one from the right-hand circle. These indicate two identical daughter duplexes that each have an outer blue circle around an inner red circle.

Determination of whether the replication loops originate at a unique point in the DNA required landmarks along the DNA molecule. These landmarks were provided by a technique called denaturation mapping, developed by Ross Inman and colleagues. Using the 48,502 bp chromosome of bacteriophage $λ$ $lamda$ , Inman showed that DNA could be selectively denatured at sequences unusually rich in $A ═ T$ $upper A box drawings double horizontal upper T$ base pairs, generating a reproducible pattern of single-strand bubbles (see Fig. 8-28). Using the denatured regions as points of reference, investigators have subsequently been able to measure the position and progress of the replication forks. Inman and colleagues found that the replication loops always initiated at a unique point, which was termed an origin. They also confirmed the earlier observation that replication is usually bidirectional. For circular DNA molecules, the two replication forks meet at a point on the side of the circle opposite to the origin. Specific origins of replication have since been identified and characterized in bacteria and eukaryotes.

DNA Synthesis Proceeds in a $5^{'} \to 3^{'}$ $bold 5 prime right-arrow bold 3 prime$ Direction and Is Semidiscontinuous

A new strand of DNA is always synthesized in the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ direction, with the free $3' -OH$ $3 prime hyphen OH$ as the point at which the DNA is elongated. (Recall from Chapter 8 that the $5'$ $5 prime$ end lacks a nucleotide attached to the $5'$ $5 prime$ position, and the $3'$ $3 prime$ end lacks a nucleotide attached to the $3'$ $3 prime$ position.) Because the two DNA strands are antiparallel, the strand serving as the template is read from its $3'$ $3 prime$ end toward its $5'$ $5 prime$ end.

If synthesis always proceeds in the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ direction, how can both strands be synthesized simultaneously? If both strands were synthesized continuously while the replication fork moved, one strand would have to undergo $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ synthesis. This problem was resolved by Reiji Okazaki and colleagues in the 1960s. Okazaki found that one of the new DNA strands is synthesized in short pieces, now called Okazaki fragments. Thus, one strand is synthesized continuously and the other discontinuously (Fig. 25-2). The continuous strand, or leading strand, is the one for which $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ synthesis proceeds in the same direction that the replication fork moves. The discontinuous strand, or lagging strand, is the one in which $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ synthesis proceeds in the direction opposite to the direction of fork movement. Okazaki fragments are typically 150 to 200 nucleotides long in eukaryotes, and 1,000 to 2,000 nucleotides long in bacteria. As we shall see, leading and lagging strand syntheses are tightly coordinated.

A figure shows the details of D N A strands at a replication fork. — FIGURE 25-2 Defining DNA strands at the replication fork. A new DNA strand (light red) is always synthesized in the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ direction. The template is read in the opposite direction, $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ . The leading strand is continuously synthesized in the direction taken by the replication fork. The other strand, the lagging strand, is synthesized discontinuously in short pieces (Okazaki fragments) in a direction opposite to that in which the replication fork moves. The Okazaki fragments are spliced together by DNA ligase. In bacteria, Okazaki fragments are ∼1,000 to 2,000 nucleotides long. In eukaryotic cells, they are 150 to 200 nucleotides long.

On the right, two blue strands of D N A run parallel. The top strand ends at its 5 prime end and the bottom strand ends at its 3 prime end. To the left, about halfway across the illustration, the two strands separate with the top strand angling diagonally upward to its 3 prime end at the upper left and the bottom strand angling diagonally downward to its 5 prime end at the lower left. A large red arrow points into the fork created by the separation of these strands and is labeled, direction of movement of replication fork. The top blue D N A strand is labeled leading strand. Beneath its 3 prime end of the blue strand at the upper left, there is the 5 prime end of a red arrow that runs beneath it to reach an arrowhead labeled 3 prime near the fork where the two blue strands separate. The bottom blue strand is labeled lagging strand. Above the 5 prime end of the blue stand at the lower left, the 3 prime end of a red strand labeled Okazaki fragment runs a short distance to its 5 prime end about halfway along the diagonal piece of this lower blue strand. To its right, a red arrow representing a second Okazaki fragment has its 5 prime end near the replication fork and runs to the right to its 3 prime end right end, an arrowhead, that is a short distance before the 5 prime end of the first Okazaki fragment.

DNA Is Degraded by Nucleases

To explain the enzymology of DNA replication, we first introduce the enzymes that degrade DNA rather than synthesize it. These enzymes are known as nucleases, or DNases if they are specific for DNA rather than RNA. Every cell contains several different nucleases, belonging to two broad classes: exonucleases and endonucleases. Exonucleases degrade nucleic acids from one end of the molecule. Many operate in only the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ direction or the $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ direction, removing nucleotides only from the $5'$ $5 prime$ end or the $3'$ $3 prime$ end, respectively, of one strand of a double-stranded nucleic acid or of a single-stranded DNA. Endonucleases can begin to degrade at specific internal sites in a nucleic acid strand or molecule, reducing it to smaller and smaller fragments. A few exonucleases and endonucleases degrade only single-stranded DNA. A few important classes of endonucleases cleave only at specific nucleotide sequences (such as the restriction endonucleases that are so important in biotechnology; see Chapter 9, Fig. 9-2). You will encounter many types of nucleases in this and subsequent chapters.

DNA Is Synthesized by DNA Polymerases

A photo shows Arthur Kornberg in a white lab coat holding a piece of equipment and standing in front of a bench with equipment on it. — Arthur Kornberg, 1918–2007

The search for an enzyme that could synthesize DNA began in 1955. Work by Arthur Kornberg and colleagues led to the purification and characterization of a DNA polymerase from E. coli cells, a single-polypeptide enzyme now called DNA polymerase I ( $M_{r} 103,000$ $upper M Subscript r Baseline 103,000$ ; encoded by the polA gene). Much later, investigators found that E. coli contains at least four additional distinct DNA polymerases, described below.

Detailed studies of DNA polymerase I revealed features of the DNA synthetic process that are now known to be common to all DNA polymerases. The fundamental reaction is a phosphoryl group transfer. The nucleophile is the $3'$ $3 prime$ -hydroxyl group of the nucleotide at the $3'$ $3 prime$ end of the growing strand. Nucleophilic attack occurs at the α phosphorus of the incoming deoxynucleoside $5'$ $5 prime$ -triphosphate (Fig. 25-3). Inorganic pyrophosphate is released in the reaction. The general reaction is

\begin{matrix} \underset{DNA}{{(dNMP)}_{n}} + dNTP & \to & \underset{Lengthened DNA}{{(dNMP)}_{n + 1}} \end{matrix} + {PP}_{i}

$StartLayout 1st Row 1st Column left-parenthesis dNMP right-parenthesis Subscript n Baseline Underscript DNA Endscripts plus dNTP 2nd Column right-arrow 3rd Column left-parenthesis dNMP right-parenthesis Subscript n plus 1 Baseline Underscript Lengthened DNA Endscripts EndLayout plus PP Subscript i$

(25-1)

where dNMP and dNTP are a deoxynucleoside $5'$ $5 prime$ -monophosphate and $5'$ $5 prime$ -triphosphate, respectively. Catalysis by virtually all DNA polymerases prominently involves two ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ions at the active site. One of these helps to deprotonate the $3'$ $3 prime$ -hydroxyl group, rendering it a more effective nucleophile. The other binds to the incoming dNTP and facilitates departure of the pyrophosphate.

A figure shows the D N A polymerase reaction. — MECHANISM FIGURE 25-3 The DNA polymerase reaction. The catalytic mechanism for addition of a new nucleotide by DNA polymerase involves two ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate, the $3'$ $3 prime$ -hydroxyl group that will act as a nucleophile, and three Asp residues, two of which are highly conserved in all DNA polymerases. The ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ion depicted at the top facilitates attack of the $3'$ $3 prime$ -hydroxyl group of the primer on the α phosphate of the nucleotide triphosphate; the other ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ion facilitates displacement of the pyrophosphate. Both ions stabilize the structure of the pentacovalent transition state. RNA polymerases use a similar mechanism.

MECHANISM FIGURE 25-3 The DNA polymerase reaction. The catalytic mechanism for addition of a new nucleotide by DNA polymerase involves two ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate, the $3'$ $3 prime$ -hydroxyl group that will act as a nucleophile, and three Asp residues, two of which are highly conserved in all DNA polymerases. The ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ion depicted at the top facilitates attack of the $3'$ $3 prime$ -hydroxyl group of the primer on the α phosphate of the nucleotide triphosphate; the other ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ion facilitates displacement of the pyrophosphate. Both ions stabilize the structure of the pentacovalent transition state. RNA polymerases use a similar mechanism.

D N A polymerase is shown as a yellow roughly rectangular structure on the left with two rounded invaginations and a rounded lower right side. Each invagination contains a green sphere labeled M g 2 plus. In D N A polymerase, A s p is bonded to O above and below with a dashed curved line running between the two O atoms with a negative symbol in a circle located next to the curve at the fork between the bonds running to the two O atoms. Dashed lines extend from the upper O to the upper M g 2 plus ion and from the bottom O to the lower M g 2 plus ion. To the right of the first A s p, there is a second A s p bonded to two O atoms with a dashed curved line between them and a negative charge in a circle just to the right of the dashed line at the fork. Dashed lines extend from the top O to the upper M g 2 plus and from the bottom O to the bottom M g s 2 plus. Two vertical stands of D N A are shown to the right. The left-hand strand is labeled growing strand (primer) and the right-hand strand is labeled template strand. The template strand is blue except for the nitrogenous basses and the growing strand is red except for the nitrogenous bases. From top to bottom, the template strand begins with the label 3 prime connected by a dashed line to the upper right vertex of a five-carbon sugar with its adjacent lower right vertex bonded to C H 2 bonded to O bonded to the upper right vertex of a second five-carbon sugar. This pattern continues to join seven five-carbon sugars in total ending with a dashed line from the lower right vertex of the bottom sugar to the label 5 prime. Each sugar has a bond from its lower left vertex to a nitrogenous base. The top sugar is bonded to the lower right vertex of a red hexagon labeled C that is connected by three hydrogen bonds, each represented by three blue short vertical lines, to a green base labeled G on the growing strand. These bonds connect from a bond from the upper left vertex of C to a bond from the upper right vertex of G, from the left side vertex of C to the left side vertex of G, and from a bond from the lower left vertex of C to a bond at the lower right vertex of G. The second sugar is bonded from the lower left vertex to the lower left vertex of a blue five-carbon sugar that shares its left side with the upper right side of a six-membered ring labeled A to the right. Hydrogen bonds connect a bond from the upper left vertex of A to a bond from the upper right vertex of a yellow base labeled T from the template strand and the left side vertex of A to the right side vertex of T. The third sugar is connected by its lower left vertex to the lower left vertex of a green double-ring base labeled G that resembles A and that is connected by hydrogen bonds from a top bond from the upper left vertex to a bond from the upper right vertex of a red six-carbon ring labeled C of the growing strand, from the left vertex of G to the right side vertex of C, and from a bond from the lower left vertex of G to a bond from the lower right vertex of C. The fourth sugar of the template strand is connected by its lower left vertex to the upper right left vertex of a red six-membered ring labeled C with bonds extending from its upper and lower left vertices. The fifth sugar of the template strand is connected by its lower left vertex to the lower left vertex of a blue five-membered ring with its left side joined to a right side labeled A with a bond extending from its upper left vertex. The sixth sugar of the template strand is connected by its lower left vertex to the lower left vertex of a green five-membered ring that shares its left side with the upper right side of a six-membered ring labeled G with bonds extending from its upper and lower left vertices. From top to bottom, the growing strand (primer) begins with the label 5 prime connected by a dashed line to C H 2 further bonded to the upper left vertex of a five-carbon sugar with its adjacent upper right vertex bonded to the lower left vertex of a nitrogenous base and its lower left vertex bonded to O bonded to C H 2 bonded to to the upper right vertex of a second five-carbon sugar. This pattern continues to join three five-carbon sugars in total ending with a a bond from the lower left vertex of the bottom sugar to O with a red negative sign labeled 3 prime. Each base is connected by hydrogen bonds to bases in the template strand as previously described. From top to bottom, the top sugar is bonded to a green double ring labeled G, the second sugar is bonded to a yellow single ring labeled T, and the third sugar is bonded to a red single ring labeled C. A dashed line extends from the top M g 2 plus to the red negative symbol of O bonded to the lower left vertex of the bottom red sugar of the growing strand, from which a red arrow points to P to the lower left. This P is labeled alpha and is bonded to four substituents. To the right, this P is bonded to O to the right further bonded to C H 2 bonded to the upper left vertex of a five-membered ring that has a lower left vertex labeled 3 prime bonded to O H and an upper right vertex labeled 5 prime bonded to the lower left vertex of a blue ring that shares its lower right side with the upper right side of a blue six-membered ring labeled A that is connected by dashed hydrogen bonds by an upper bond at the upper right vertex to an upper bond at the upper left vertex of T of the template strand and with a bond from it right side to the left side bond of T. This nucleotide is labeled incoming d N T P. The P is also bonded to O minus above, O O below further bonded to P beta below double bonded to O to the right, bonded to O minus to the left connected by a dashed line to the bottom M g minus, and bonded to O below further bonded to P gamma double bonded to O to the right, bonded to O minus below, and bonded too minus to the left connected by a dashed bond to the bottom M g 2 plus. P alpha also has a double bond to the left that is connected by dashed bonds to the M g 2 plus ions to the upper and lower left. A red arrow points from the bond between P alpha and O below to the same O below.

The reaction seems to proceed with only a minimal change in free energy, given that one phosphodiester bond is formed at the expense of a somewhat less stable phosphate anhydride. However, noncovalent base-stacking and base-pairing interactions provide additional stabilization to the lengthened DNA product relative to the free nucleotide. Also, the formation of products is facilitated in the cell by the 19 kJ/mol generated in the subsequent hydrolysis of the pyrophosphate product by the enzyme pyrophosphatase (p. 485).

Early work on DNA polymerase I led to the definition of two central requirements for DNA polymerization (Fig. 25-4). First, all DNA polymerases require a template. The polymerization reaction is guided by a template DNA strand according to the base-pairing rules predicted by Watson and Crick: where a guanine is present in the template, a cytosine deoxynucleotide is added to the new strand, and so on. This was a particularly important discovery, not only because it provided a chemical basis for accurate semiconservative DNA replication, but also because it represented the first example of the use of a template to guide a biosynthetic reaction.

A three-part figure illustrates the elongation of a D N A chain showing the structure of D N A polymerase Roman numeral 1 interacting with D N A in part a, the structure of the core of a D N A polymerase in part b, and a cartoon interpretation of the insertion site of D N A polymerase in part c. — FIGURE 25-4 Elongation of a DNA chain. (a) DNA polymerase I activity requires a single unpaired strand to act as template and a primer strand to provide the free hydroxyl group at the $3'$ $3 prime$ end to which the new nucleotide unit is added. Each incoming complementary nucleotide is bound selectively, in part by base-pairing to the appropriate nucleotide in the template strand. The reaction product has a new free $3'$ $3 prime$ hydroxyl, allowing the addition of another nucleotide. The newly formed base pair translocates to make the active site available to the next pair to be formed. (b) The core of most DNA polymerases is shaped like a human hand that wraps around the active site. The structure shown here is DNA polymerase I of *Thermus aquaticus*, bound to DNA. (c) A cartoon interpretation shows the insertion site, where the nucleotide addition occurs, and the postinsertion site, to which the newly formed base pair translocates. [(b) Data from PDB ID 4KTQ, Y. Li et al., *EMBO J.* 17:7514, 1998.]

Part A shows yellow Pol Roman numeral 1 as a half-circle with an opening in its bottom center, resembling an inverted “U” shape. Two strands of D N A are shown. The top red strand is labeled primer strand. It begins outside of the polymerase as 5 prime G A A, reaches the edge of Pol Roman numeral 1 at T, and continues with C G T before C enclosed in a vertical orange oval labeled primer terminus with a curved line above and a similar green oval to the right. A red A labeled incoming d N T P is shown approaching the opening in the green oval. Beneath, a blue complementary strand begins at its 3 prime end with C beneath G of the primer stand. The template strand is 3 prime C T T, then reaches the edge of Pol Roman numeral 1 at A, then continues as G C A before reaching G in the bottom of the orange oval labeled postinsertion, T in the bottom of the green oval labeled insertion site, and then G C A A T 5 prime at the end of the polymerase molecule. The opening at the bottom of the polymerase is beneath the orange and green ovals. An arrow points right beneath the word insertion. This yields a similar structure in which the red A labeled incoming d N T P is now in the top of the green oval above T in the template strand. An arrow labeled translocation points right to indicate a similar structure in which the polymerase has shifted right with respect to the strands. A that had been inserted into the green oval is now in the red oval to the left above T below. G is now in the bottom of the green oval and the top of the green oval is empty. Part b shows a surface contour model with a roughly triangular shape with a dark brown top part labeled palm, a gray bottom boundary beneath the dark brown region above a central opening, a yellow roughly triangular piece of the left labeled thumb that widens toward the bottom and a light brown oval piece that extends down on the right labeled fingers. An opening between the bottom of the yellow and brown parts widens to a wider region beneath the brown top piece. A piece of D N A is shown extending from the upper left of the yellow piece into the opening, where it curves against the gray part and then down along the left side of the light brown vertical piece. Part c shows a similar yellow structure to part a except that the rounded top is labeled palm, the protrusion to the lower left is labeled thumb, and the protrusion to the lower right is thicker and labeled fingers. A double helix of D N A runs horizontally from the left with a blue 3 prime strand beginning on the top and a red five prime strand beginning on the bottom. A line beneath the word palm and above an orange oval labeled postinsertion site to the left and a green oval labeled insertion site to the right is shown above an opening between the pieces labeled thumb and fingers. The red strand ends at its 3 prime end beneath the green oval labeled insertion site. The blue strand curves upward at this point and extends diagonally up to the right across the fingers portion to end at its 5 prime end to the upper right.

Second, the polymerases require a primer. A primer is a strand segment (complementary to the template) with a free $3'$ $3 prime$ -hydroxyl group to which a nucleotide can be added; the free $3'$ $3 prime$ end of the primer is called the primer terminus (Fig. 25-4a). In other words, part of the new strand must already be in place: all DNA polymerases can add nucleotides only to a preexisting strand. Many primers are oligonucleotides of RNA rather than DNA, and specialized enzymes synthesize primers when and where they are required.

A DNA polymerase active site has two parts (Fig. 25-4a). The incoming nucleotide is initially positioned in the insertion site. Once the phosphodiester bond is formed, the polymerase slides forward on the DNA and the new base pair is positioned in the postinsertion site. These sites are located in a pocket that resembles the palm of a hand (Fig. 25-4b, c).

After adding a nucleotide to a growing DNA strand, a DNA polymerase either dissociates or moves along the template and adds another nucleotide. Dissociation and reassociation of the polymerase can limit the overall polymerization rate — the process is generally faster when a polymerase adds more nucleotides without dissociating from the template. The average number of nucleotides added before a polymerase dissociates defines its processivity. DNA polymerases vary greatly in processivity; some add just a few nucleotides before dissociating, whereas others add many thousands.

Replication Is Very Accurate

Replication proceeds with an extraordinary degree of fidelity. In E. coli, a mistake is made only once for every $10^{9}$ $10 Superscript 9$ to $10^{10}$ $10 Superscript 10$ nucleotides added. For the E. coli chromosome of $\sim 4.6 \times 10^{6}$ $tilde 4.6 times 10 Superscript 6$ bp, this means that an error occurs only once per 1,000 to 10,000 replications. During polymerization, discrimination between correct and incorrect nucleotides relies not just on the hydrogen bonds that specify the correct pairing between complementary bases but also on the common geometry of the standard $A ═ T$ $upper A box drawings double horizontal upper T$ and $G \equiv C$ $upper G identical-to upper C$ base pairs (Fig. 25-5). The active site of DNA polymerase I accommodates only base pairs with this geometry. An incorrect nucleotide may be able to hydrogen-bond with a base in the template, but it generally will not fit into the active site. Incorrect bases can be rejected before the phosphodiester bond is formed.

A figure shows how base-pair geometry contributes to the fidelity of D N A replication with part a showing how the geometries of the standard base pairs fit into active sites and part b showing how incorrectly paired bases can be excluded from the active site. — FIGURE 25-5 Contribution of base-pair geometry to the fidelity of DNA replication. (a) The standard $A ═ T$ $upper A box drawings double horizontal upper T$ and $G \equiv C$ $upper G identical-to upper C$ base pairs have very similar geometries, and an active site sized to fit one will generally accommodate the other. (b) The geometry of incorrectly paired bases can exclude them from the active site, as occurs on DNA polymerase.

FIGURE 25-5 Contribution of base-pair geometry to the fidelity of DNA replication. (a) The standard $A ═ T$ $upper A box drawings double horizontal upper T$ and $G \equiv C$ $upper G identical-to upper C$ base pairs have very similar geometries, and an active site sized to fit one will generally accommodate the other. (b) The geometry of incorrectly paired bases can exclude them from the active site, as occurs on DNA polymerase.

Part a is labeled correct base pairs. The top oval shown C with three hydrogen bonds to G and the bottom ovals shows T with two hydrogen bonds to A, both fitting into the openings. A yellow oval contains an open region in the center with narrow entries to the lower left and right and a wider center. The narrow entry at the lower left contains three dots that lead to N substituted for C at the lower left vertex of a red six-membered ring labeled C with double bonds at the upper left and right sides, N substituted for C at the right side vertex, a double bond to O from the lower right vertex, and a bond to N from the upper right vertex that is further bonded to 2 H. The narrow entry at the lower right contains three dots that lead to N substituted for C at the bottom vertex of a green five-membered ring with a double bond at its upper right side, N substituted for C at the top vertex, and a double bond at its left side shared with the right side of a six-membered ring labeled C that has a double bond at its lower left side, N substituted for C at its lower right vertex, a lower left vertex bonded to N further bonded to 2 H, N substituted for C at the left side and bonded to H, and an upper left vertex double bonded to O. There are hydrogen bonds represented by three short vertical blue bars between H bonded to N bonded to the upper right vertex of C and O double bonded to the upper left of G, between N at the left side of C and H bonded to N at the right side of the six-membered ring of G, and O double bonded to the lower right vertex of C to H bonded to N bonded to the lower right vertex of the six-membered ring of G. A similar structure is shown below with a yellow six-membered ring labeled T on the left and a blue double-ring labeled A on the right. The structures are similar except that the yellow T has N at its right side bonded to H, an upper right vertex double bonded to O, and an upper left vertex bonded to C H 3, and no double bond at the upper right side. The blue double-ring structure labeled A is similar to G except that there is a double bond at the upper left side, N at the left side is not bonded to H, and the upper left vertex is bonded to N bonded to 2 H. There are hydrogen bonds between O double bonded to the upper right vertex of T and H bonded to N bonded to the upper left vertex of the six-membered ring of A and H bonded to N at the right side of T to A at the right side of the six-membered ring of A. Part b is labeled incorrect base pairs. Three ovals are shown with one showing T bonded to G, one showing C bonded to A, and one showing A bonded to G. These did not fit into the openings within the ovals. The upper left oval has T at the upper right and G at the right side. T and G have the same shapes as previously described. There are hydrogen bonds between H bonded to N at the right side of T and O double boned to the upper left vertex of G and between O double boned to the lower right vertex of T and H bonded to N at the right side of G. T is pushed against the upper left corner of the oval. Tot eh right, an oval contains A on the left oriented with the six-membered ring above and the give-membered ring below and pushed against the upper left side. It also has G on the right side. There are hydrogen bonds between H bonded to N bonded to the right side vertex of the six-membered ring of A and O double bonded to the upper left vertex of G and from N at the lower right vertex of the five-membered ring of A to H bonded to N at the right side vertex of G. The lower left oval shows C on the left and A on the right with A pushed against the lower right corner. There are double bonds between N at the right side of C and H bonded to N bonded to the upper left vertex of the six-membered ring of A and between O double bonded to the lower right vertex of C and H bonded to N at the right side of the six-membered ring of A.

The accuracy of the polymerization reaction itself, however, is insufficient to account for the high degree of fidelity in replication. Careful measurements in vitro have shown that DNA polymerases insert one incorrect nucleotide for every $10^{4} to 10^{5}$ $10 Superscript 4 Baseline to 10 Superscript 5$ correct ones. These mistakes sometimes occur because a base is briefly in an unusual tautomeric form (see Fig. 8-9), allowing it to hydrogen-bond with an incorrect partner. In vivo, the error rate is reduced by additional enzymatic mechanisms.

One mechanism intrinsic to many DNA polymerases is a separate $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ exonuclease activity that double-checks each nucleotide after it is added. This nuclease activity permits the enzyme to remove a newly added nucleotide and is highly specific for mismatched base pairs (Fig. 25-6). If the polymerase has added the wrong nucleotide, translocation of the enzyme to the position where the next nucleotide is to be added is inhibited. This kinetic pause provides the opportunity for a correction. The $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ exonuclease activity cleaves the most recently added phosphodiester bond and removes the mispaired nucleotide; the polymerase then adds another nucleotide to begin active synthesis again. This activity, known as proofreading, is not simply the reverse of the polymerization reaction (Eqn 25-1). Instead, replacement of the incorrect nucleotide requires the expenditure of three high-energy bonds. The polymerizing and proofreading activities of a DNA polymerase can be measured separately. Proofreading improves the inherent accuracy of the polymerization reaction $10^{2} - to 10^{3} - fold$ $10 squared hyphen to 10 cubed zero width space hyphen fold$ . In the monomeric DNA polymerase I, the polymerizing and proofreading activities have separate active sites within the same polypeptide.

A figure shows an example of error correction by the 3 prime to 5 prime exonuclease activity of D N A polymerase Roman numeral 1 in five steps. — FIGURE 25-6 An example of error correction by the $3' \to 5'$ $bold 3 prime bold right-arrow bold 5 prime$ exonuclease activity of DNA polymerase I. Structural analysis has located the exonuclease activity behind the polymerase activity as the enzyme is oriented in its movement along the DNA. A mismatched base (here, a C–T mismatch) impedes translocation of DNA polymerase I (Pol I) to the next site.

FIGURE 25-6 An example of error correction by the $3' \to 5'$ $bold 3 prime bold right-arrow bold 5 prime$ exonuclease activity of DNA polymerase I. Structural analysis has located the exonuclease activity behind the polymerase activity as the enzyme is oriented in its movement along the DNA. A mismatched base (here, a C–T mismatch) impedes translocation of DNA polymerase I (Pol I) to the next site.

Step 1: polymerase mispairs d C with d T. A yellow polymerase is shown with a wide top and protrusions to the lower left and right. A red top strand begins at 5 prime to the left and runs from G A A T C C T to G in the vertical orange oval and C in the vertical green oval at the 3 prime end. The blue complementary strand begins below as 3 prime C T T A G G A with C in the orange oval, T in the green oval, and then G A C G A C T A ending at the 5 prime end. The orange vertical oval is labeled postinsertion side and the green vertical oval is labeled insertion side. An orange square labeled exonuclease site is shown above C of the red strand right before T next to G in the orange oval. An arrow points right to Step 2: Polymerase repositions to mispaired 3 prime terminus into the 3 prime to 5 prime exonuclease site. An arrow points down to show a similar structure in which the polymerase has been angled so that the ends points to the lower left. The red piece is now positioned horizontally to end above and to the left of the orange oval. It is 5 prime G A A T C C T G and then C in the red exonuclease site. Beneath, the complementary strand is 3 prime C T T A G G A C with T beneath C in the exonuclease site, then G A C G A C T A 5 prime. The vertical orange oval and vertical green oval extend diagonally from the center of the polymerase beneath the blue strand to the lower left. Step 3: Exonuclease hydrolyzes the mispaired d C. An arrow points down accompanied by a branched arrow showing the loss of d C M P. This yields a similar product in which the exonuclease site is empty because C has been removed. Step 4: The 3 prime terminus repositions back to the polymerase site. The polymerase reorients so that it looks like the illustration in step 1 except that there is no nucleotide in the top of the green oval. Step 5: Polymerase incorporates the correct nucleotide, d A. An arrow points down accompanied by a curved arrow showing the addition of d A T P. This yields a product similar to the illustration in step 1 except that A is in the top position in the green oval instead of C.

When base selection and proofreading are combined, DNA polymerase leaves behind one net error for every $10^{6} to 10^{8}$ $10 Superscript 6 Baseline to 10 Superscript 8$ bases added. Yet the measured accuracy of replication in E. coli is higher still. The additional accuracy is provided by a separate enzyme system that repairs the mismatched base pairs remaining after replication. We describe this mismatch repair, along with other DNA repair processes, in Section 25.2.

E. coli Has at Least Five DNA Polymerases

More than 90% of the DNA polymerase activity observed in E. coli extracts can be accounted for by DNA polymerase I. Soon after the isolation of this enzyme in 1955, however, evidence began to accumulate that it is not suited for replication of the large E. coli chromosome. First, the rate at which it adds nucleotides (600 nucleotides/min) is too slow (by a factor of 100 or more) to account for the rates at which the replication fork moves in the bacterial cell. Second, DNA polymerase I has a relatively low processivity. Third, genetic studies have demonstrated that many genes, and therefore many proteins, are involved in replication: DNA polymerase I clearly does not act alone. Fourth, and most important, in 1969 John Cairns isolated a bacterial strain with an altered gene for DNA polymerase I that produced an inactive enzyme. Although this strain was abnormally sensitive to agents that damaged DNA, it was nevertheless viable.

A search for other DNA polymerases led to the discovery of E. coli DNA polymerase II and DNA polymerase III in the early 1970s. DNA polymerase II is an enzyme involved in one type of DNA repair (Section 25.3). DNA polymerase III is the principal replication enzyme in E. coli. DNA polymerases IV and V, identified in 1999, are involved in an unusual form of DNA repair (Section 25.2). The properties of these five DNA polymerases are compared in Table 25-1.

TABLE 25-1 Comparison of the Five DNA Polymerases of *E. coli*
	DNA polymerase
		I	II^a	III	IV^a	V^a
Structural gene^b		polA	polB	polC (dnaE)	dinB	umuC
Subunits (number of different types)		1	7	9	1	3
$M_{r}$ $upper M Subscript r$		103,000	88,000^c	1,065,400	39,100	110,000
$3' \to 5'$ $3 prime right-arrow 5 prime$ exonuclease (proofreading)		Yes	Yes	Yes	No	No
$5' \to 3'$ $5 prime right-arrow 3 prime$ exonuclease		Yes	No	No	No	No
Polymerization rate (nucleotides/s)		10–20	40	250–1,000	2–3	1
Processivity (nucleotides added before polymerase dissociates)		3–200	1,500	≥500,000	1	6–8
^aTranslesion (mutagenic) DNA polymerases. For DNA polymerase IV, processivity is increased substantially by association with a β clamp. These polymerases are slowed when a DNA lesion is present in the DNA template strand. ^bFor enzymes with more than one subunit, the gene listed here encodes the subunit with polymerization activity. Note that dnaE is an earlier designation for the gene now referred to as polC. ^cPolymerization subunit only. DNA polymerase II shares several subunits with DNA polymerase III, including the β, $δ$ $delta$ , $δ'$ $delta prime$ , $χ$ $chi$ , and $ψ$ $psi$ subunits (see Table 25-2).

DNA polymerase I, then, is not the primary enzyme of replication; instead, it performs a host of cleanup functions during replication, recombination, and repair. The polymerase’s special functions are enhanced by its $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease activity. This activity, distinct from the $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ proofreading exonuclease (Fig. 25-6), is located in a structural domain that can be separated from the rest of the enzyme by mild protease treatment. When the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease domain is removed, the remaining fragment $(M_{r} 68, 000)$ $left-parenthesis upper M Subscript r Baseline 68 comma 000 right-parenthesis$ , the large fragment or Klenow fragment, retains the polymerization and proofreading activities. The $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease activity of intact DNA polymerase I can replace a segment of DNA (or RNA) paired to the template strand, in a process known as nick translation (Fig. 25-7). Most other DNA polymerases lack a $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease activity.

A figure shows how nick translation is used to move a break or nick in D N A along with the enzyme to aid in D N A repair and in the removal of R N A primers. — FIGURE 25-7 Nick translation. The bacterial DNA polymerase I has three domains, catalyzing its DNA polymerase, $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease, and $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ exonuclease activities. The $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease domain is in front of the enzyme as it moves along the DNA and is not shown in Figure 25-4. By degrading the DNA strand ahead of the enzyme and synthesizing a new strand behind, DNA polymerase I can promote nick translation, in which a break or nick in the DNA is effectively moved along with the enzyme. This process has a role in DNA repair and in the removal of RNA primers during replication (both described later in this chapter). The strand of nucleic acid to be removed (either DNA or RNA) is shown in purple, the replacement strand in red. DNA synthesis begins at a nick (a broken phosphodiester bond, leaving a free $3'$ $3 prime$ hydroxyl and a free $5'$ $5 prime$ phosphate). A nick remains where DNA polymerase I eventually dissociates, and the nick is later sealed by another enzyme.

A yellow Pol Roman numeral 1 is wide at the top with two arms extending to the lower left and right. In the center, there is a vertical orange oval labeled postinsertion site next to a vertical green oval labeled insertion site. An orange square labeled 3 prime to 5 prime exonuclease is shown just to the upper left of the orange oval. A yellow triangle labeled 5 prime to 3 prime exonuclease overlaps the right side of the polymerase and extends to the right. Two horizontal blue strands of D N A run from the left into the enter of the polymerase. The top strand begins at 5 prime and ends just to the right of the green oval, where there is a nick and then a purple strand in the 5 prime to 3 prime exonuclease labeled nucleic acid to be removed that ends with a blue piece that ends at its 3 prime end to the upper right. The bottom blue strand begins at its 3 prime end and bends beneath the nick in the top strand to run up to end at its 5 prime end at the upper right. An arrow points down. This indicates a similar structure in which three light red rectangles are shown on the left with an arrow pointing to the vertical green oval accompanied by text reading d N T P s added and P P subscript i end subscript released. Additionally, three purple rectangles are shown with an arrow indicating that they are leaving the left side of the 5 prime to 3 prime exonuclease, where the left end of the purple piece of d N A Is missing. Text reads, n M P s or d N M P s release. A gray arrow points right labeled, Pol Roman numeral 1 movement. An arrow points down with a curved line showing the addition of d N T P s and a branched arrow showing the loss of N M P s or d N M P s, P P subscript i end subscript i. This yields a similar structure in which the top strand begins at the blue 5 prime end and reaches a pink region that runs across the vertical orange oval and halfway across the vertical green oval. There is a space, then a blue diagonal piece runs up to end at the 3 prime end to the upper right. The blue bottom strand begins at its 3 prime end to the left and runs beneath the top strand, continuing across the vertical green oval and then bending upward to end at the upper right at its 3 prime end.

DNA polymerase III is much more complex than DNA polymerase I, with nine different kinds of subunits (Table 25-2). Its polymerization and proofreading activities reside in its α and ε subunits, respectively. The $θ$ $theta$ subunit associates with α and ε to form a core polymerase, which can polymerize DNA but with limited processivity. Up to three core polymerases can be linked by a clamp-loading complex consisting of five subunits of three different types, $τ_{3} δ δ'$ $tau 3 delta delta prime$ . The core polymerases are linked through the $τ$ $tau$ (tau) subunits. Two additional subunits, $χ$ $chi$ (chi) and $ψ$ $psi$ (psi), are bound to the clamp-loading complex. The entire assembly of 16 protein subunits (eight different types) is called DNA polymerase III* (Fig. 25-8a).

TABLE 25-2 Subunits of DNA Polymerase III of *E. coli*

The table consists of 5 columns and 9 rows. Column headings: Subunit; Number of subunits per holoenzyme; M subscript r of subunit; Gene; Function of subunit. Row 1: Alpha; 3; 129,900; p o l C (d n a E); Polymerization activity. Row 2: Epsilon; 3; 27,500; d n a Q (m u t D); 3 prime to 5 prime proofreading exonuclease. Row 3: Theta; 3; 8,600; h o l E; Stabilization of epsilon subunit. Row 4: Tau; 3; 71,100; d n a X; Stable template binding, core enzyme dimerization. Row 5: Delta; 1; 38,700; h o l A; Clamp opener. Row 6: Delta prime; 1; 36,900; h o l B; Clamp loader. Row 7: Chi; 1; 16,600; h o l C; Interaction with S S B. Row 8: Psi; 1; 15,200; h o l D; Interaction with Tau and Chi. Row 9: Beta; 6; 40,600; d n a N; D N A clamp required for optimal processivity. Table notes indicate that Alpha, Epsilon, and Theta are part of Core polymerase. Tau, Delta, and Delta prime are part of Clamp-loading (Gamma) complex that loads Beta subunits on lagging strand at each Okazaki fragment. A footnote here reads: The Gamma subunit is encoded by a portion of the gene for the Tau subunit (d n a X), such that the amino-terminal 66 percent of the Tau subunit has the same amino acid sequence as the Gamma subunit. The Gamma subunit is generated by a translational frameshifting mechanism (page 1013) that leads to premature translational termination. The Gamma subunit shares the clamp-loading functions of Tau but lacks the protein segments that interact with the core polymerase or with D n a B helicase. Clamp-loading complexes incorporating Tau subunits may operate independently of the D N A polymerase I II holoenzyme, promoting the unloading of Beta clamps discarded on the lagging strand as the replication fork progresses. They may also promote loading of Beta clamps for some D N A repair processes that require D N A synthesis away from the replication fork.

A two-part figure shows the structure of bacterial D N A polymerase Roman numeral 3 as an illustration in part a and as ribbon and surface contour images in part b. — FIGURE 25-8 DNA polymerase III. (a) Architecture of bacterial DNA polymerase III (Pol III). Three core domains, composed of subunits α, ε, and $θ$ $theta$ , are linked by a five-subunit clamp-loading complex, with the composition $τ_{3} δ δ'$ $tau 3 delta delta prime$ . The core subunits and clamp-loading complex constitute DNA polymerase III*. The other two subunits of DNA polymerase III*, $χ$ $chi$ and $ψ$ $psi$ (not shown), also bind to the clamp-loading complex. Three β clamps interact with the three core subassemblies, each clamp a dimer of the β subunit. The complex interacts with the DnaB helicase (described later in the text) through the $τ$ $tau$ subunits. (b) Two β subunits of *E. coli* polymerase III form a circular clamp that surrounds the DNA. The clamp slides along the DNA molecule, increasing the processivity of the polymerase III holoenzyme to more than 500,000 nucleotides by preventing its dissociation from the DNA. The two β subunits are shown in two shades of purple as ribbon structures (left) and surface contour images (right), surrounding the DNA. [(a) Information from N. Yao and M. O’Donnell, *Mol. Biosyst.* 4:1075, 2008. (b) Data from PDB ID 2POL, X.-P. Kong et al., *Cell* 69:425, 1992.]

Part a shows a ring of five pieces labeled clamp loader at the bottom. Each piece has a rounded top that curves to a thinner tail. Two bright green pieces at the front are labeled delta prime and delta on the left and right, respectively. The remaining three pieces are labeled tau. The two side pieces are light green and the piece in the rear is more cylindrical and dark green. Each tau piece has a green strand extending up to a medium green piece labeled tau that is rounded where it meets the strand than then has a thinner curved pieces. The left-hand piece meets a curved yellow bar that I part of the Pol Roman numeral 3 core that runs at a slight angle from the left to the lower right, where it curves toward the viewer. Across its left side, there is a crescent-shaped bar labeled Pol Roman numeral 3 core. Above the top of the Pol Roman numeral 3 core, there is a dark purple half circle beta sliding clamp adjacent to a light purple piece representing the other half with a round opening between them. The central tau has a strand to a similar green piece that meets a yellow bar that extends up and bends toward the viewer below with a pol Roman numeral 3 core across the top and the beta sliding clamp to its right vertically positioned and angled slightly to the right. The third tau has a strand to a green piece to the right that meets a yellow bar that runs to the right and bends toward the viewer to the left with a crescent-shaped bar running vertically along the right side and the beta sliding clamp positioned horizontally below. The yellow Pol 3 core crescents meet the beta sliding clamps at their dark purple halves. Part b shows a rung with an open center that has dark purple on the top half and light purple on the bottom half. There are helices along the border of the central opening and beta strands and sheets around the outside. A strand of D N A is visible extending away from the viewer in the open center. To its right, a surface contour image of the same thing shows a circle with a dark purple top, a light purple bottom, and an open center containing a strand of D N A extending away from the viewer.

DNA polymerase III* can polymerize DNA, but with a much lower processivity than one would expect for the organized replication of an entire chromosome. The necessary increase in processivity is provided by the addition of the β subunits. The β subunits associate in pairs to form donut-shaped structures that encircle the DNA and act like clamps (Fig. 25-8b). Each dimer associates with a core subassembly of polymerase III* (one dimeric clamp per active core subassembly) and slides along the DNA as replication proceeds. The β sliding clamp prevents the dissociation of DNA polymerase III from DNA, dramatically increasing processivity — to greater than 500,000 (Table 25-1). The addition of the β subunits converts DNA polymerase III* to DNA polymerase III holoenzyme.

DNA Replication Requires Many Enzymes and Protein Factors

Replication in E. coli requires not just a single DNA polymerase but 20 or more different enzymes and proteins, each performing a specific task. The entire complex has been termed the DNA replicase system or replisome. The enzymatic complexity of replication reflects the constraints imposed by the structure of DNA and by the requirements for accuracy. The main classes of replication enzymes are considered here in terms of the problems they overcome.

The DNA must be separated into two strands that each act as a template. This is generally accomplished by helicases, enzymes that move along the DNA and separate the strands, using chemical energy from ATP. Strand separation creates topological stress in the helical DNA structure (see Fig. 24-11), which is relieved by the action of topoisomerases. The separated strands are stabilized by DNA-binding proteins. As noted earlier, before DNA polymerases can begin synthesizing DNA, primers must be present on the template — generally, short segments of RNA synthesized by enzymes known as primases. Ultimately, the RNA primers are removed and replaced by DNA; in E. coli, this is one of the many functions of DNA polymerase I. A specialized nuclease that degrades RNA in RNA-DNA hybrids, called RNase H1, also removes some RNA primers. After an RNA primer is removed and the gap is filled in with DNA, a nick remains in the DNA backbone in the form of a broken phosphodiester bond. These nicks are sealed by DNA ligases. All these processes require coordination and regulation best characterized in the E. coli system.

Replication of the E. coli Chromosome Proceeds in Stages

The synthesis of a DNA molecule can be divided into three stages: initiation, elongation, and termination, distinguished both by the reactions taking place and by the enzymes required. As you will find here and in the next two chapters, synthesis of the major information-containing biological polymers — DNAs, RNAs, and proteins — can be understood in terms of these same three stages, with the stages of each pathway having unique characteristics. The events described below reflect information derived primarily from in vitro experiments using purified E. coli proteins, although the principles are highly conserved in all replication systems.

Initiation

The E. coli replication origin, oriC, consists of 245 bp and contains DNA sequence elements that are highly conserved among bacterial replication origins. The general arrangement of the conserved sequences is illustrated in Figure 25-9. Two types of sequences are of special interest: five repeats of a 9 bp sequence (R sites) that serve as binding sites for the key initiator protein, DnaA, and a region rich in $A ═ T$ $upper A box drawings double horizontal upper T$ base pairs called the DNA unwinding element (DUE). There are three additional DnaA-binding sites (I sites), and binding sites for the proteins IHF (integration host factor) and FIS (factor for inversion stimulation). These two proteins were discovered as necessary components of certain recombination reactions described later in this chapter, and their names reflect those roles. Another DNA-binding protein, HU (a histonelike bacterial protein originally dubbed factor U), also participates but does not have a specific binding site.

A figure shows the arrangement of sequences in the italicized E. coli end italics replication origin, italicized ori C end italics. — FIGURE 25-9 Arrangement of sequences in the *E. coli* replication origin, *oriC*. Conserved sequences for key repeated elements are shown. N represents any of the four nucleotides. The horizontal arrows indicate the orientations of the nucleotide sequences (left-to-right arrow denotes a sequence in the top strand; right-to-left, in the bottom strand). FIS and IHF are binding sites for proteins described in the text. R sites are bound by DnaA. I sites are additional DnaA-binding sites (with different sequences, labeled I1, I2, and I3), for DnaA only when the protein is complexed with ATP.

A horizontal bar contains man segments. On the left, it begins with a series of three orange segments that each contain an arrow pointing to the right. Text above reads, tandem array of three 13-b p sequences, consensus sequence G A T C T N T T N T T T T. An arrow branches into three arrows that point to each of the three pieces with D U E above the ventral piece. The remaining sequences are as follows: a gray piece; a small blue piece labeled R 1 with an arrow pointing to the left; a gray piece; a longer yellow piece labeled I H F; a small blue piece labeled R 5 with an arrow pointing to the left; a gray piece, a dark gray piece labeled I 1; a narrow gray piece; a ark gray piece labeled I 2; a gray piece; a blue piece labeled R 2 with an arrow pointing right; a gray piece, a longer yellow piece labeled F I S; a gray piece; a blue piece labeled R 3 with an arrow pointing left; a gray piece; a dark gray piece labeled I 3; a gray piece; a blue piece labeled R 4 with an arrow pointing to the right; and a gray piece. Text above reads, binding sites for D n a A protein, five 9-b p sequences, consensus sequence T T (A / T) T N C A C C has an arrow pointing down that points to the five blue pieces with arrows: R 1, R 5, R 2, R 3, and R 4.

At least 10 different enzymes or proteins (summarized in Table 25-3) participate in the initiation phase of replication. They open the DNA helix at the origin and establish a prepriming complex for subsequent reactions. The crucial component in the initiation process is the DnaA protein, a member of the $AAA + ATPase$ $bold AAA zero width space bold plus bold ATPase$ protein family (ATPases associated with diverse cellular activities). Many $AAA + ATPases$ $AAA zero width space plus ATPases$ , including DnaA, form oligomers and hydrolyze ATP relatively slowly. This ATP hydrolysis acts as a switch that mediates interconversion of the protein between two states. In the case of DnaA, the ATP-bound form is active and the ADP-bound form is inactive.

TABLE 25-3 Proteins Required to Initiate Replication at the *E. coli* Origin
Protein	$M_{r}$ $bold-italic upper M Subscript bold r$	Number of subunits	Function
DnaA protein	52,000	1	Recognizes oriC sequence; opens duplex at specific sites in origin
DnaB protein (helicase)	300,000	6^a	Unwinds DNA
DnaC protein	174,000	6^a	Required for DnaB binding at origin
HU	19,000	2	Histonelike protein; DNA-binding protein; stimulates initiation
FIS	22,500	2^a	DNA-binding protein; stimulates initiation
IHF	22,000	2	DNA-binding protein; stimulates initiation
Primase (DnaG protein)	60,000	1	Synthesizes RNA primers
Single-stranded DNA–binding protein (SSB)	75,600	4^a	Binds single-stranded DNA
DNA gyrase (DNA topoisomerase II)	400,000	4	Relieves torsional strain generated by DNA unwinding
Dam methylase	32,000	1	Methylates $(5^{'})$ $left-parenthesis 5 prime right-parenthesis$ GATC sequences at oriC
^aSubunits in these cases are identical.

Eight DnaA protein molecules, all in the ATP-bound state, assemble to form a helical complex encompassing the R and I sites in oriC (Fig. 25-10). DnaA has a higher affinity for R sites than I sites, and it binds R sites equally well in its ATP- or ADP-bound form. The I sites, which bind only the ATP-bound DnaA, allow discrimination between the active and inactive forms of DnaA. The tight right-handed wrapping of the DNA around this complex introduces a positive supercoil (see Chapter 24). The associated strain in the nearby DNA, combined with the binding of additional DnaA protein to the DUE region, leads to strand separation in the $A ═ T - rich$ $upper A box drawings double horizontal upper T hyphen rich$ DUE. The complex formed at the replication origin also includes several DNA-binding proteins — $HU, IHF,$ $HU comma IHF comma$ and $FIS$ $FIS$ — that facilitate DNA bending.

A figure shows a model for initiation of replication at the italicized E. coli end italics origin, italicized ori C end italics. — FIGURE 25-10 Model for initiation of replication at the *E. coli* origin, *oriC*. DnaA protein molecules bind initially at the five specific R sites. Upon ATP binding, additional DnaA molecules bind at the I sites in the origin, forming a right-handed helical complex and drawing in more DnaA molecules that continue the helix into the DUE region (see Fig. 25-9). The DNA is wrapped around this complex. DnaA molecules bound at the R and I sites bind the DNA with an HTH doman (referring to a DNA binding motif called helix-turn-helix). The $A ═ T$ $upper A box drawings double horizontal upper T$ -rich DUE region is denatured as a result of the strain imparted by the DnaA binding. Within the DUE, single-stranded DNA is bound by the ATPase domain of DnaA rather than by the HTH. Formation of the helical DnaA complex is facilitated by the proteins HU, IHF, and FIS. The detailed structural roles of these proteins are not known, but IHF may stabilize a transient DNA loop, as shown here. Hexamers of the DnaB protein bind to each strand, with the aid of DnaC protein. The DnaB helicase activity further unwinds the DNA in preparation for priming and DNA synthesis. [Information from J. P. Erzberger et al., *Nat. Struct. Mol. Biol.* 13:676, 2006.]

A long strand with several bends begin with a blue piece, bends left through a short gray piece, then becomes an orange piece labeled D U E that curves up and meets a gray piece that bends down to a series of two purple spheres that join to a gray piece that ends with a shorter blue piece. The region from the orange piece labeled D U E to the gray piece on the right, excluding the blue pieces, is labeled ori C. The purple spheres are labeled D n a A dash A A A plus. An arrow points down next to a gray box labeled origin binding. This yields a structure with a blue horizontal piece that meets a short gray piece that meets an orange loop that meets a gray piece that runs down, becomes black, and loops back across itself to run up to the right to form an upward curve before becoming gray again and running down to end with an almost horizontal blue piece. There is a chain of purple spheres that begins just to the right to the start of the orange ring and bends down to the top of the gray loop, then runs up and down again beneath the upward gray loop to end at the start of the blue piece. The left-hand chain of purple spheres has alternating green partial triangles that lack bases and green spheres running across its top. One of these green spheres is labeled D n a A H T H. Where the gray piece loops down, two beige vertical cylinders extend down to beige spheres labeled D n a A H T H. A similar sphere is visible to the right and above the purple spheres where they loop up. At the top of the right-hand loop, two of these cylinders extend up to beige spheres that meet the top of the gray loop. Two more beige spheres are visible along the last two purple spheres. At the bottom of the black and gray downward loop, there are two gray vertical ovals labeled I H F. Accompanying text reads, D n a A-dependent denaturation of the A T-rich D U E region. An arrow points down accompanied by text reading D n a C-dependent loading of D N a B helicase. This yields a similar structure except that the orange bubble has a ring of dark blue spheres labeled D n a B near its center left connected by strands to light blue spheres labeled D n a C immediately before the chain of purple spheres begins farther to the right. A similar set of dark and alight blue spheres is visible at the lower right of the orange bubble with the light blue spheres on the left and the dark blue spheres on the right. The black and gray loop extending down no longer contains the two spheres labeled I H F.

The DnaC protein, another $AAA + ATPase$ $AAA zero width space plus ATPase$ , then loads the DnaB protein onto the separated DNA strands in the denatured region. A hexamer of DnaC, each subunit bound to ATP, forms a tight complex with the ring-shaped, hexameric DnaB helicase. This DnaC-DnaB interaction opens the DnaB ring, the process being aided by a further interaction between DnaB and DnaA. Two of the ring-shaped DnaB hexamers are loaded in the DUE, one onto each DNA strand. The ATP bound to DnaC is hydrolyzed, releasing the DnaC and leaving the DnaB bound to the DNA.

Loading of the DnaB helicase is the key step in replication initiation. As a replicative helicase, DnaB migrates along the single-stranded DNA in the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ direction, unwinding the DNA as it travels. The DnaB helicases loaded onto the two DNA strands thus travel in opposite directions, creating two potential replication forks. All other proteins at the replication fork are linked directly or indirectly to DnaB. The DNA polymerase III holoenzyme is linked through its $τ$ $tau$ subunits; additional DnaB interactions are described below. As replication begins and the DNA strands are separated at the fork, many molecules of single-stranded DNA–binding protein (SSB) bind to and stabilize the separated strands, and DNA gyrase (DNA topoisomerase II) relieves the topological stress induced ahead of the fork by the unwinding reaction.

Initiation is the only phase of DNA replication that is known to be regulated, and it is regulated such that replication occurs only once in each cell cycle. The mechanism of regulation is not yet entirely understood, but genetic and biochemical studies have provided insights into several separate regulatory mechanisms.

Once DNA polymerase III has been loaded onto the DNA, along with the β subunits (signaling completion of the initiation phase), the protein Hda binds to the β subunits and interacts with DnaA to stimulate hydrolysis of its bound ATP. Hda is yet another $AAA + ATPase$ $AAA zero width space plus ATPase$ closely related to DnaA (its name is derived from homologous to DnaA). This ATP hydrolysis leads to disassembly of the DnaA complex at the origin. Slow release of ADP by DnaA and rebinding of ATP cycles the protein between its inactive (with bound ADP) and active (with bound ATP) forms on a time scale of 20 to 40 minutes.

The timing of replication initiation is affected by DNA methylation and interactions with the bacterial plasma membrane. The oriC DNA is methylated by the Dam methylase (Table 25-3), which methylates the $N^{6}$ $upper N Superscript 6$ position of adenine within the palindromic sequence $(5')$ $left-parenthesis 5 prime right-parenthesis$ GATC. (Dam is not a biochemical expletive; it stands for DNA adenine methylation.) The oriC region of E. coli is highly enriched in GATC sequences — it has 11 in its 245 bp; the average frequency of GATC in the E. coli chromosome as a whole is 1 in 256 bp.

Immediately after replication, the DNA is hemimethylated: the parent strands have methylated oriC sequences but the newly synthesized strands do not. The hemimethylated oriC sequences are now sequestered by interaction with the plasma membrane (the mechanism is unknown) and by binding of the protein SeqA. After a time, oriC is released from the plasma membrane, SeqA dissociates, and the DNA must be fully methylated by Dam methylase before it can again bind DnaA and initiate a new round of replication.

Elongation

The elongation phase of replication includes two distinct but related operations: leading strand synthesis and lagging strand synthesis. Several enzymes at the replication fork are important to the synthesis of both strands. Parent DNA is first unwound by DNA helicases, and the resulting topological stress is relieved by topoisomerases. Each separated strand is then stabilized by SSB. From this point, synthesis of leading and lagging strands is sharply different.

Leading strand synthesis, the more straightforward of the two, begins with the synthesis by primase (DnaG protein) of a short (10 to 60 nucleotide) RNA primer at the replication origin. DnaG interacts with DnaB helicase to carry out this reaction, and the primer is synthesized in the direction opposite to that in which the DnaB helicase is moving. In effect, the DnaB helicase moves along the strand that becomes the lagging strand in DNA synthesis; however, the first primer laid down in the first DnaG-DnaB interaction serves to prime leading strand DNA synthesis in the opposite direction. Deoxyribonucleotides are added to this primer by a DNA polymerase III complex linked to the DnaB helicase tethered to the opposite DNA strand. Leading strand synthesis then proceeds continuously, keeping pace with the unwinding of DNA at the replication fork.

Lagging strand synthesis, as we have noted, is accomplished in short Okazaki fragments (Fig. 25-11a). First, an RNA primer is synthesized by primase, and, as in leading strand synthesis, DNA polymerase III binds to the RNA primer and adds deoxyribonucleotides (Fig. 25-11b). On this level, the synthesis of each Okazaki fragment seems straightforward, but the details are quite complex. The complexity lies in the coordination of leading and lagging strand synthesis. Both strands are produced by a single asymmetric DNA polymerase III dimer; this is accomplished by looping the DNA of the lagging strand as shown in Figure 25-12, bringing together the two points of polymerization.

A two-part figure shows how Okazaki fragments are synthesized with part a showing the structures involved and part b showing the replisome complex. — FIGURE 25-11 Synthesis of Okazaki fragments. (a) At intervals, primase synthesizes an RNA primer for a new Okazaki fragment. If we consider the two template strands as lying side by side, lagging strand synthesis formally proceeds in the opposite direction from fork movement. Each primer is extended by DNA polymerase III. DNA synthesis continues until the fragment extends as far as the primer of the previously added Okazaki fragment. A new primer is synthesized near the replication fork to begin the process again. (b) In the replisome complex, DNA synthesis on the leading and lagging strands is tightly coordinated. Each DNA polymerase III holoenzyme has three sets of core subunits (yellow), linked together with a single clamp-loading complex, so one or two Okazaki fragments can be synthesized simultaneously, along with the leading strand.

Part a shows two parallel blue horizontal strands of D N A. The top strand begins at its 5 prime end at the bottom half begins at its 3 prime end. A “C” shaped structure curve around the back of the two stands and is labeled D N S topoisomerase Roman numeral 2 (D N A gyrase). A red arrow above pointing right is labeled replication form movement. To the right, the two strands separate so that the top strand is higher and the bottom strand is lower. Near the left end of the raised part of the top strand, P o l Roman numeral 3 is shown with a horizontal bar that curves toward the viewer beneath the strand and has a crescent across the top. The blue strand runs right to end at its 3 prime end. Above it, a red strand begins at its 5 prime end on the left and ends at an arrow within Pol Roman numeral 3. At the right of the lower strand, as it bends down, there is a circle of dark blue spheres labeled D n a B helicase around the blue strand. To its right, there is a vertical orange var with horizontal bars across the top and bottom labeled primase with a short green bar inside just beneath the blue strand that is labeled R N A rimer. TO the right, there are three sets of four teal spheres each, with two at the top of the blue strand and two at the bottom of the strand. These are labeled S S B. To the right, Pol Roman numeral 3 is shown as a vertical bar that bends toward the viewer across the top of the blue stand and ha a crescent shape below the strand. To its left, a strand has a short green piece joined to a red arrow that ends in Pol Roman numeral 3. This is labeled lagging strand synthesis (Okazaki fragment). A set of four S S B are to the right. To the right of this, there is another Okazaki fragment with a short green piece joined to a longer red piece that ends at its 3 prime end beneath the 5 prime end of the blue strand above. Part b shows two horizontal bleu strands beneath a red arrow pointing to the left. To the right, this separates into upper and lower halves. The upper part has a Pol Roman numeral 3 with a vertical piece that runs across the blue strand with a crescent-shaped core at the top and a piece that curves toward the viewer at the bottom. A purple ring is to its right with a dark purple top half and a light purple bottom half. The blue strand extends to the right and a red strand runs above it to end at an arrow in the middle of Pol Roma numeral 3. The strand bending downward runs through a ring of dark blue spheres, then bends left, then bends back to the right through a purple ring that is light on top and dark on the bottom adjacent to a Pol Roman numeral 3 with its crescent part labeled core toward the bottom. The blue stand runs through this, curves left, then curves back toward the right. As the blue strand runs up through another purple sphere with its dark side to the right, across a Pol Roman numeral 3 with its crescent-shaped core to the lower right, and then runs horizontally to the right. A red strand runs horizontally beneath this blue strand until just before the Pol Roman numeral 3, where it ha a small green piece. A red arrow ends in the core and begins with a small green piece fot he lower left. A green structure with three projections to the right is labeled clamp-loading complex has is connected to each of the three Pol 3 pieces.

A five-part figure shows D N A synthesis on the leading and lagging strands in five stages. — FIGURE 25-12 DNA synthesis on the leading and lagging strands. Events at the replication fork are coordinated by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. This figure shows the replication process already underway; (a) through (e) are discussed in the text. Only two sets of polymerase core subunits rather than three are shown, to more clearly illustrate the cycling on the lagging strand. The lagging strand is looped so that DNA synthesis proceeds steadily on both the leading and lagging strand templates at the same time. Red arrows indicate the $3'$ $3 prime$ end of the two new strands and the direction of DNA synthesis. An Okazaki fragment is being synthesized on the lagging strand. The subunit colors and the functions of the clamp-loading complex are explained in Figure 25-13.

Part a shows a blue strand of D N A beneath a horizontal piece of red D N A to the right. The red piece is labeled lagging strand and ends with a small green piece on the left labeled R N A primer of previous Okazaki fragment. The blue strand continues to run through a D N A pol Roman numeral 3 with a crescent-shaped piece at the bottom, a vertical piece running behind the blue strand, and a third piece curving above the blue strand toward the viewer. A vertical purple circle to the left has a light purple piece on top and a dark purple piece below with the dark purple piece aligned with the left end of the crescent-shaped piece of D N A pol Roman numeral 3. The blue strand runs through the open center of this purple center with a red piece of D N A running parallel on top that has a small green piece on its left end and an arrow at its right end within D N A pol Roman numeral 3. The blue strand curves up, to the right, and then left through a ring of six blue spheres. One of these blue spheres is labeled D n a B. A green piece connects the lower right piece of this ring to the top of the D N A pol Roman numeral 3 below and a small thread extends from its upper half to a light green piece of a structure to the right that forms part of a partial circle of green structures. A second green piece connects the upper right of the ring of blue spheres to the bottom of an R N A Roman numeral 3 above and has a strand connecting to a light green piece in the same right-hand structure. This right-hand structure has a dark green rectangular piece across the bottom with the light green pieces to its upper and lower sides and toward the front. The upper light green piece is beneath a dark green piece with a rounded left end that curves downward and a thinner right end that ends at the upper right end of a dark purple half circle that loops down to the left along the light green and dark green pieces of the top half of the overall structure. The lower light green piece is beneath a similar dark green piece with a rounded left end that extends upward and that ends at the top left of a light purple half circle that loops down along the bottom half of the structure, forming an overall ring of green pieces that align with the purple ring to the right side and with no piece across the top. This structure is labeled, clamp-loading complex with open beta sliding clamp. The blue strand that passed through the ring of dark blue spheres to the left continues left and meets a parallel blue strand beneath a large red arrow pointing left. The top blue strand bends upward to the right and runs across the vertical center of a yellow D N A pol Roman numeral 3 that curves toward the viewer below and has a crescent-shaped piece labeled core above. A purple ring is vertically aligned with Roman pol 3 with its upper purple half touching the right end of the upper crescent of R N A pol 3. The blue strand runs through the center of this circle. A red strand labeled leading strand runs beneath the blue strand at the upper right and through the circle to end at an arrow halfway across D N A pol 3. A gray box of accompanying text reads, Continuous synthesis on the leading strand proceeds as D N A is unwound by the D n a B helicase. An arrow points down to part b accompanied by a curved line showing the addition of a brown rectangle curved toward the viewer at each end and labeled primase. This yields a similar structure in which primase is inserted beneath the ring of six blue spheres so that the blue strand of D N A runs through it with a small green parallel piece labeled new R N A primer adjacent to the blue strand within the primase. The primase ends behind the light blue top half of a purple circle next to D N A pol 3 below. The red strand above the blue strand at the right end is now longer and extends almost to the bottom D N A pol Roman numeral 3 before ending at a small green piece. A gray box above this strand reads, Okazaki fragment synthesis nears completion. A gray box below reads, primase binds to D n a B, synthesizes a new primer, then dissociates. An arrow points down accompanied by a branched arrow showing the loss of primase. This yields structure c, which is a similar structure in which a clamp-loading complex is shown centered between the upper and lower D N A pol Roman numeral 3. Three blue spheres of the previous ring of blue spheres are visible to the left, a dark purple half ring is at the top front, and a light purple ring is at the bottom front. A light green part is visible behind the dark purple half ring, a light green part is visible behind the light purple half ring, and a dark green horizontal piece is shown along the right that meets the purple ring where its right halves meet. The blue strand of D N A curves into the center of the purple ring, where a curved green strand is aligned with it, then continues out to the left beneath the three blue spheres before joining a second blue strand. A dashed arrow points to the dark green piece at the side of the green structure and is accompanied by text in a gray box reading, A new beta clamp is loading onto the new template primer by the clamp loader. A gray box beneath the strand of D N A to the right beneath a red strand with a green left end reads, Synthesis of a new Okazaki fragment is completed on the lagging strand. An arrow points right to structure d, which is similar except for the following differences. The right-hand piece of blue D N A now has blue on top above a red piece with a small green piece to the left on the far right. Slightly to the left, another red strand runs through a purple ring that is light purple on top and dark purple on top and is labeled, beta clamp left behind. This red strand ends with a small green piece as the blue piece above curves down. The blue strand loops up and through the bottom D N A pol Roman numeral 3, which it exists through a purple ring with the light purple piece on top. A small green strand begins in D N A pol Roman numeral 3 and extends to the purple ring. A dashed arrow points from just before the purple ring labeled beta clamp left behind to the center of the bottom D N A pol Roman numeral 3. The blue strand continues out from the purple ring and loops up, then to the right to run through a ring of six blue spheres before becoming horizontal beneath a second blue strand above beneath a red arrow pointing to the left. The ring of blue spheres has two green pieces extending from it to the upper and lower D N A pol Roman numeral 3. Each green piece has a strand extending to a green piece of a clamp-loading complex with a dark green horizontal piece at the back, green pieces above and below that each are narrow on the right and wider to the left, and light green pieces at the top and bottom. A dashed arrow points from the bottom green pieces that connects the ring of blue spheres to the D N A pol Roman numeral 3 below to the green piece at the bottom center of the clamp-loading complex. Accompanying text in a gray box reads, Lagging strand core subunits are transferred to the new primer template and its beta clamp. The older beta clamp is left behind. An arrow points up to point e which is similar to part a. Accompanying text reads, The next beta clamp is readied as Okazaki fragment synthesis is initiated.

The synthesis of Okazaki fragments on the lagging strand entails some elegant enzymatic choreography. DNA polymerase III uses one set of its core subunits (the core polymerase) to synthesize the leading strand continuously, while the other two sets of core subunits cycle from one Okazaki fragment to the next on the looped lagging strand. In vitro, a DNA polymerase III holoenzyme with only two sets of core subunits can synthesize both leading and lagging strands. However, a third set of core subunits increases the efficiency of lagging strand synthesis as well as the processivity of the overall replisome.

DnaB helicase, bound in front of DNA polymerase III, unwinds the DNA at the replication fork (Fig. 25-12a) as it travels along the lagging strand template in the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ direction. DnaG primase occasionally associates with DnaB helicase and synthesizes a short RNA primer (Fig. 25-12b). A new β sliding clamp is then positioned at the primer by the clamp-loading complex of DNA polymerase III (Fig. 25-12c). When synthesis of an Okazaki fragment has been completed, replication halts, and the core subunits of DNA polymerase III dissociate from their β sliding clamp (and from the completed Okazaki fragment) and associate with the new clamp (Fig. 25-12d, e). This initiates synthesis of a new Okazaki fragment. Two sets of core subunits may be engaged in the synthesis of two different Okazaki fragments at the same time. The proteins acting at the replication fork are summarized in Table 25-4.

TABLE 25-4 Proteins of the *E. coli* Replisome
Protein	$M_{r}$ $bold-italic upper M Subscript bold r$	Number of subunits	Function
SSB	75,600	4	Binding to single-stranded DNA
Helicase (DnaB protein)	300,000	6	DNA unwinding
Primase (DnaG protein)	60,000	1	RNA primer synthesis
DNA polymerase III	1,065,400	17	New strand elongation
DNA polymerase I	103,000	1	Filling of gaps; excision of primers
DNA ligase	74,000	1	Ligation
DNA gyrase (DNA topoisomerase II)	400,000	4	Supercoiling

The clamp-loading complex of DNA polymerase III, consisting of parts of the three $τ$ $tau$ subunits along with the $δ$ $delta$ and $δ'$ $delta prime$ subunits, is also an $AAA + ATPase$ $AAA zero width space plus ATPase$ . This complex binds to ATP and to the new β sliding clamp. The binding imparts strain on the dimeric clamp, opening up the ring at one subunit interface (Fig. 25-13). The newly primed lagging strand is slipped into the ring through the resulting break. The clamp loader then hydrolyzes ATP, releasing the β sliding clamp and allowing it to close around the DNA.

A figure shows the structure of the D N A polymerase Roman numeral 3 clamp loader. — FIGURE 25-13 The DNA polymerase III clamp loader. The five subunits of the clamp-loading $(γ)$ $left-parenthesis gamma right-parenthesis$ complex are the $δ$ $delta$ and $δ'$ $delta prime$ subunits and the amino-terminal domain of each of the three $τ$ $tau$ subunits (see Fig. 25-8). The complex binds to three molecules of ATP and to a dimeric β clamp. This binding forces the β clamp open at one of its two subunit interfaces. Hydrolysis of the bound ATP allows the β clamp to close again around the DNA.

FIGURE 25-13 The DNA polymerase III clamp loader. The five subunits of the clamp-loading $(γ)$ $left-parenthesis gamma right-parenthesis$ complex are the $δ$ $delta$ and $δ'$ $delta prime$ subunits and the amino-terminal domain of each of the three $τ$ $tau$ subunits (see Fig. 25-8). The complex binds to three molecules of ATP and to a dimeric β clamp. This binding forces the β clamp open at one of its two subunit interfaces. Hydrolysis of the bound ATP allows the β clamp to close again around the DNA.

A strand of blue D N A begins at the 3 prime end of the lower left and curves up rapidly, then more gently to a 5 prime end at the upper right. A small green strand runs horizontally above the blue strand just beneath a large green structure. This structure has a light green vertical piece at the lower right with a rounded left end connected to A T P and connected by a green strand to a curved green piece that ends at a light purple sphere labeled A T P just above the strand. Above this light green vertical piece, there is a bright green horizontal piece that is flat on top, has a rounded left end that extends inward, and has a narrower right end that joins with the upper right side of light purple half circle. A dark horizontal piece across the bottom has a line to the purple sphere labeled A T P at the left and joins with the lower right of a dark purple half circle to the right labeled beta-clamp. A light green piece to the upper left of this dark green piece Is similar to the other light green piece but horizontal. It is bonded to A T P at its left end and is connected by a strand to a similar green piece that runs upward from the light purple sphere labeled A T P. To the upper right of this light green piece, there is a dark green piece labeled clamp loader that is vertical, rounded to the left so that it protrudes inward, and narrow to the right until it ends at the upper part of the dark purple beta clamp. An arrow points right to show a similar structure with a blue strand of D N A running through the center across the lower green piece beneath the light purple sphere labeled A T P, across the top of the curved part of the horizontal bright green piece, and out through an opening at the upper right between the dark and light purple parks of the beta clamp. A green parallel strand begins just beneath the dark large green structure formed by the light and dark green pieces. An arrow points right accompanied by a branched arrow showing the loss of the entire green structure with all of the A T P molecules changed to A D P. 3 P i are also lost. This yields a vertical ring with a dark purple top and light purple bottom with a blue stand running through it from a 3 prime end at the upper left to a 5 prime end at the lower right. A small green strand extends from the purple ring to run a short distance to the left above the blue strand.

The replisome promotes rapid DNA synthesis, adding ~1,000 to 2,000 nucleotides to each strand (leading and lagging). Once an Okazaki fragment has been completed, its RNA primer is removed by DNA polymerase I or RNase H1 and replaced with DNA by the polymerase; the remaining nick is sealed by DNA ligase (Fig. 25-14).

A figure shows the final steps in the synthesis of lagging strand segments. — FIGURE 25-14 Final steps in the synthesis of lagging strand segments. RNA primers in the lagging strand are removed by the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease activity of DNA polymerase I or RNase H1, and then replaced with DNA by DNA polymerase I. The remaining nick is sealed by DNA ligase. The role of ATP or ${NAD}^{+}$ $NAD Superscript plus$ is shown in Figure 25-15.

A blue strand of D N A runs from its 3 prime end on the left to its 5 prime end on the right, where it is labeled lagging strand. A red strand below begins beneath the 3 prime end of the blue strand as a red arrow that points right to a space labeled nick about one-quarter of the length across the strand. A green piece to the right is about the same length and joins a red strand that runs to end at its 3 prime end beneath the 5 prime end of the blue strand above. An arrow labeled blue highlighted D N A polymerase Roman numeral 1 or blue highlighted R N ase H 1 is accompanied by a branched arrow showing the loss of N M P s and a curved line showing the addition of d N T Ps. This yields a solid blue line above a red line below that consists of an arrow that ends at an arrowhead pointing right to the place where the green piece ended, from which a solid red line continues to the right to end beneath the right end of the blue strand. An arrow labeled blue highlighted D N A ligase points down accompanied by a curved arrow showing the addition of A T P (or N A D plus) and loss of A M P plus P P subscript I end subscript (or N M N). This yields a solid blue line above a solid red line.

DNA ligase catalyzes the formation of a phosphodiester bond between a $3'$ $3 prime$ hydroxyl at the end of one DNA strand and a $5'$ $5 prime$ phosphate at the end of another strand. The phosphate must be activated by adenylylation. DNA ligases isolated from viruses and eukaryotes use ATP for this purpose. DNA ligases from bacteria are unusual in that many use ${NAD}^{+}$ $NAD Superscript plus$ — a cofactor that usually functions in hydride transfer reactions (see Fig. 13-24) — as the source of the AMP activating group (Fig. 25-15). DNA ligase is another enzyme of DNA metabolism that has become an important reagent in recombinant DNA experiments (see Fig. 9-1).

A figure shows the mechanism of the D N A ligase reaction in three steps. — FIGURE 25-15 Mechanism of the DNA ligase reaction. In each of the three steps, one phosphodiester bond is formed at the expense of another. Steps and lead to activation of the $5'$ $5 prime$ phosphate in the nick. An AMP group is transferred first to a Lys residue on the enzyme and then to the $5'$ $5 prime$ phosphate in the nick. In step , the $3'$ $3 prime$ -hydroxyl group attacks this phosphate and displaces AMP, producing a phosphodiester bond to seal the nick. In the *E. coli* DNA ligase reaction, AMP is derived from ${NAD}^{+}$ $NAD Superscript plus$ . The DNA ligases isolated from some viral and eukaryotic sources use ATP rather than ${NAD}^{+},$ $NAD Superscript plus Baseline comma$ and they release pyrophosphate rather than nicotinamide mononucleotide (NMN) in step .

FIGURE 25-15 Mechanism of the DNA ligase reaction. In each of the three steps, one phosphodiester bond is formed at the expense of another. Steps and lead to activation of the $5'$ $5 prime$ phosphate in the nick. An AMP group is transferred first to a Lys residue on the enzyme and then to the $5'$ $5 prime$ phosphate in the nick. In step , the $3'$ $3 prime$ -hydroxyl group attacks this phosphate and displaces AMP, producing a phosphodiester bond to seal the nick. In the *E. coli* DNA ligase reaction, AMP is derived from ${NAD}^{+}$ $NAD Superscript plus$ . The DNA ligases isolated from some viral and eukaryotic sources use ATP rather than ${NAD}^{+},$ $NAD Superscript plus Baseline comma$ and they release pyrophosphate rather than nicotinamide mononucleotide (NMN) in step .

A blue oval labeled enzyme is bonded to N H 3 with a positive charge on N. This is labeled D N A ligase. Step 1: Adenylation of D N A ligase. Upward- and downward-pointing arrows indicate a reversible reaction. A curved arrow meets the downward arrow. The curved arrow shows the addition of R bonded to O bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the right further bonded to a red box labeled ribose bonded to a blue box labeled adenine, labeled A M P from A T P (R equals P P subscript i end subscript) or N A D plus (R equals N M N), and the loss of P P subscript i end subscript (from A T P) or N M N (from N A D plus). This yields a blue oval labeled enzyme bonded to N H 2 with a positive charge on N bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the right bonded to a light red box labeled ribose bonded to a light blue box labeled adenine. Text below reads, enzyme – A M P. Step 2: Activation of 5 prime phosphate in nick. A piece of D N A is shown as two strands. The top strand that runs from the 3 prime end on the left to the 5 prime end on the right. Four vertical lines extend down on the left and four vertical lines extend down on the right, leaving an open region in the middle. The bottom strand begins at the 5 prime end on the lower left and has four vertical lines extending up with the final vertical line bonded to O H. There is a space beneath the open region in the top strand, then a phosphate bonded to the left end of a strand with three upward vertical lines before reaching the five prime end. The phosphate has O bonded to the D N A strand and bonded to p to the lower left, bonded to O minus to the lower right and left, and double bonded to O to the upper right. Right- and left-pointing arrows indicate a reversible reaction. A curved arrow meets the right-pointing arrow and extends from enzyme-A M P on the left to a blue oval labeled enzyme bonded to N H 3 with a positive charge on N on the right. This yields a similar molecule in which the bottom strand has O H shown with a red pair of electrons on O and a second phosphate group has been added, meaning that the lower left end of the right-hand bottom piece of D N A is bonded to O bonded to P double bonded to O to the upper left, bonded to O minus to the lower right, and bonded to O to the lower left further bonded to P double bonded to O to the upper left, bonded to O minus to the lower left, and bonded to O to the lower right further bonded to a red box labeled ribose bonded to a blue box labeled adenine. Red arrows point from the pair of red electrons on O bonded to the lower left piece of D N A to the upper P and from the bond between the same P and O to its lower left to the same O to the lower left. Step 3: Displacement of A M P seals nick. Right- and left-pointing arrows above blue highlighted D N A ligase indicate a reversible reaction. An arrow branching from the right-pointing arrow shows the loss of P double bonded to O above, bonded to O minus to the left and below, and bonded to a blue box labeled ribose to the right further bonded to a blue box labeled adenine. The two strands of D N A are shown. The top strand is unchanged. The lower left piece now has six upward vertical pieces with the right-hand piece bonded to O bonded to P bonded to O minus below, double bonded to O above, and bonded to O to the right further bonded to the lower left of the right-hand piece of D N A, which has five upward vertical lines. Text below reads, sealed D N A.

Termination

Eventually, the two replication forks of the circular E. coli chromosome meet at a terminus region containing multiple copies of a 20 bp sequence called Ter (Fig. 25-16). The Ter sequences are arranged on the chromosome to create a trap that a replication fork can enter but cannot leave. The Ter sequences function as binding sites for the protein Tus (terminus utilization substance). The Tus-Ter complex can arrest a replication fork from only one direction. Only one Tus-Ter complex functions per replication cycle — the complex first encountered by either replication fork. Given that opposing replication forks generally halt when they collide, Ter sequences would not seem to be essential, but they may prevent overreplication by one fork in the event that the other is delayed or halted by an encounter with DNA damage or some other obstacle.

A blue circular chromosome has a vertical line labeled origin at the twelve o’clock position. An arrow pointing clockwise from the twelve o’clock to two o’clock positions is labeled clockwise fork and a similar arrow pointing counterclockwise is labeled counterclockwise fork. A region at the bottom center of the chromosome is labeled italicized T e r end italics region and runs from about halfway past the five o’clock position to about halfway past the six o’clock position. A close-up shows that this red region is divided into two halves. All of the labels are italicized and have uppercase T lowercase e lowercase r followed by an uppercase letter. Each labeled is accompanied by a rectangle with a triangular cutout. The left half of the region is labeled clockwise fork trap and all of the rectangles have triangular cutouts on the right. The right half of the red region is labeled counterclockwise fork trap and all of the rectangles have triangular cutouts on the left. From left to right, the clockwise fork trap portion of the red piece is labeled T er J at the far left, then T e r G next to T e r F, then T e r B next to T e r C. From left to right, the counterclockwise fork trap portion of the red piece is labeled T e r A next to T e r D, T e r E, and T e r I next to T e r H. All data are approximate.

So, when either replication fork encounters a functional Tus-Ter complex, it halts; the other fork halts when it meets the first (arrested) fork. The final few hundred base pairs of DNA between these large protein complexes are then replicated (by an as yet unknown mechanism), completing two topologically interlinked (catenated) circular chromosomes (Fig. 25-17). DNA circles linked in this way are known as catenanes. Separation of the catenated circles in E. coli requires topoisomerase IV (a type II topoisomerase). The separated chromosomes then segregate into daughter cells at cell division. The terminal phase of replication of other circular chromosomes, including many of the DNA viruses that infect eukaryotic cells, is similar.

A figure shows the role of topoisomerases in replication termination. — FIGURE 25-17 Role of topoisomerases in replication termination. Replication of the DNA separating opposing replication forks leaves the completed chromosomes joined as catenanes, or topologically interlinked circles. The circles are not covalently linked, but because they are interwound and each is covalently closed, they cannot be separated — except by the action of topoisomerases. In *E. coli*, a type II topoisomerase known as DNA topoisomerase IV plays the primary role in separating catenated chromosomes, transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the break.

Two circular chromosomes are shown with one above the other and a light blue region where they are joined. A close-up of this light blue region shows a blue double helix of D N A that has forks on each side. At the left side, the blue strands separate to the upper left and right and each is intertwined with a red strand. A red arrow pointing into this fork reads, counterclockwise fork. On the right, the blue strands separate to the upper right and left and each is intertwined with a red strand. A red arrow pointing into this fork reads, clockwise fork. An arrow pointing down is labeled, completion of replication. This yields a structure labeled catenated chromosomes that consists of two stands of D N A that are intertwined to produce a series of four circles. An arrow points down accompanied by text reading, blue highlighted D N A topoisomerase Roman numeral 4. This yields two separate, parallel horizontal chromosomes that each have one light blue and one red strand.

Replication in Eukaryotic Cells Is Similar but More Complex

The DNA molecules in eukaryotic cells are considerably larger than those in bacteria and are organized into complex nucleoprotein structures (chromatin; p. 898). The essential features of DNA replication are the same in eukaryotes and bacteria, and many of the protein complexes are functionally and structurally conserved. However, eukaryotic replication is regulated and coordinated with the cell cycle, and must function within the complexities of chromatin structure.

Origins of replication have a well-characterized structure in some lower eukaryotes, but they are much less defined in higher eukaryotes. In both cases, replication begins in short nucleosome-free regions. Yeast (S. cerevisiae) has about 400 defined replication origins called autonomously replicating sequences (ARSs), or replicators. Yeast replicators span ∼150 bp and contain several essential, conserved sequences. There are about 30,000 to 50,000 replication origins in human chromosomes. The replication origins of vertebrates in general may be defined by some aspect of DNA secondary structure, as yet unknown.

Regulation ensures that all cellular DNA is replicated once per cell cycle. Much of this regulation involves proteins called cyclins and the cyclin-dependent kinases (CDKs) with which they form complexes (see Section 12.8). The cyclins are rapidly destroyed by ubiquitin-dependent proteolysis at the end of the M phase (mitosis), and the absence of cyclins allows the establishment of prereplicative complexes (pre-RCs) on replication initiation sites. In rapidly growing cells, the pre-RC forms at the end of M phase. In slow-growing cells, it does not form until the end of G1. Formation of the pre-RC renders the cell competent for replication, an event sometimes called licensing.

As in bacteria, the key event in the initiation of replication in all eukaryotes is the loading of the replicative helicase, a heterohexameric complex of minichromosome maintenance (MCM) proteins (MCM2 to MCM7). The ring-shaped MCM2–7 helicase functions in some ways like the bacterial DnaB helicase, although it translocates $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ along the leading strand template. It is loaded onto the DNA in steps (Fig. 25-18). The origin is recognized and bound first by another six-protein complex, called ORC (origin recognition complex), followed by the protein CDC6 (cell division cycle), which recruits CDT1 (CDC10-dependent transcript 1). Together, they facilitate the loading of two inactive MCM2–7 complexes (the pre-RC). The ORC-CDC6 complex and CTD1 dissociate, leaving behind the pre-RC. ORC has five $AAA + ATPase$ $AAA zero width space plus ATPase$ domains among its subunits and is functionally analogous to the bacterial DnaA. The yeast CDC6 is yet another $AAA + ATPase$ $AAA zero width space plus ATPase$ that forms a complex with the ORC subunits. Following pre-RC formation, another set of proteins, CDC45 and the GINS, bind to and activate the MCM2–7 helicase, triggering DNA denaturation. (GINS refers to the first letters of the numbers 5-1-2-3 in Japanese, go-ichi-ni-san, providing a somewhat cryptic callout to the four protein subunits of the complex: SLD5, PSF1, PSF2, and PSF3.) The replication proteins then bind to form a replisome, and bidirectional replication begins.

A figure shows how a prereplicative complex is assembled at a eukaryotic replication origin. — FIGURE 25-18 Assembly of a prereplicative complex at a eukaryotic replication origin. The initiation site (origin) is bound by ORC, CDC6, and CDT1. These proteins, many of them $AAA + ATPases$ $AAA zero width space plus ATPases$ , promote loading of two MCM2–7 helicase complexes, in a reaction analogous to the loading of the bacterial DnaB helicase by DnaC protein. The two loaded but inactive MCM2–7 complexes comprise the prereplicative complex, or pre-RC. The pre-RC is subsequently activated by addition of CDC45 and the GINS proteins, followed by addition of the replisome components. [Information from M. W. Parker et al., *Crit. Rev. Biochem. Mol. Biol.* 52:107, 2017.]

FIGURE 25-18 Assembly of a prereplicative complex at a eukaryotic replication origin. The initiation site (origin) is bound by ORC, CDC6, and CDT1. These proteins, many of them $AAA + ATPases$ $AAA zero width space plus ATPases$ , promote loading of two MCM2–7 helicase complexes, in a reaction analogous to the loading of the bacterial DnaB helicase by DnaC protein. The two loaded but inactive MCM2–7 complexes comprise the prereplicative complex, or pre-RC. The pre-RC is subsequently activated by addition of CDC45 and the GINS proteins, followed by addition of the replisome components. [Information from M. W. Parker et al., *Crit. Rev. Biochem. Mol. Biol.* 52:107, 2017.]

A blue horizontal strand of D N A is shown with a small red region in the center labeled origin. A gray box above reads, The initiation site (origin) is bound by O R C and C D C 6. Below, a green complex is shown that consists of five green pieces that are all rounded at the left and narrow at the right. These form a partial circle open at the front. To the left this is labeled O R C. The top front green piece is bonded to an orange oval labeled A T P. A similarly shaped red piece below is labeled C D C 6. An arrow points up from these structures accompanied by a curved line showing the addition of an orange oval labeled A T P. This yields a green structure around a similar piece of D N A to the right of the first piece of D N A. The green structure is over a red piece of D N A that extends slightly to the left. There are two green pieces above. The front green piece is bound to an orange oval labeled A T P. A horizontal red piece runs across the D N A between two green pieces above and two green pieces below. Text below reads, O R C, C D C 6. There is a bond to A T P from upper left corner of the red piece. and the bottom of the upper front green piece meet. Above, a roughly circular complex of six horizontal blue ovals is labeled M C M - 7 and is shown just above a yellow half-circle labeled C D T 1. A gray box reads, O R C, C D C 6, and C D T 1 facilitate loading of two M C M 2 – 7 helicase complexes, which form the pre-R C. An arrow points from M C M 2 – 7 and C D T 1 to another similar piece of D N A now has a yellow half oval above the right upper side of the red horizontal piece, with its flat side facing the red piece, and a series of two M C M 2 – 7 complexes encircling the D N A to the right. Text below reads, pre-R C double hexamer. An arrow points down and branches to show the loss of the yellow half-circle, the loss of the green pieces forming a roughly circular shape, and the loss of the red bar bonded to a gray circle labeled A D P and P subscript i end subscript. An arrow shows the addition of a dark greenish brown pyramidal structure with a rounded end that points to the right and with second structure attached that has a similar base visible to the left. A purple circle with an opening in the center labeled G I N S is also added. A gray box above reads, The pre-R C is subsequently activated by addition of C D C 45 and the G I N S proteins, triggering D N A denaturation. This yields a blue pee of D N A with two M C M 2 – 7 rings of ovals around the central region. The double stranded D N A separates as it enters the structure on the left and the right. On the left, one piece of D N A disappears behind the structure and the other runs down and then between C D C 45 and the top of G I N S before running upward to pass above C D C 45 and across the lower portion of G I N S to the left of the second M C M 2 – 7 helicase. The second strand becomes visible again and the two join to the right o this complex. Text below the complex reads, pre-I C. An arrow labeled resplisome components points to double-stranded D N A that opens on its right side to form a bidirectional replication fork. A gray box above reads, Replisome components are added. A blue half-circle with flat sides is visible within the opening. Just after the top blue strand separates, it enters an M C M 2 – 7 helicase with C D C 45 on the left along the top of G I N s, an orange oval at the right end, a blue half-circle with flat sides above the orange oval, and a green oval extending to the lower right with the blue strand extending out to the right.

Commitment to replication requires the synthesis and activity of S-phase cyclin-CDK complexes (such as the cyclin E–CDK2 complex; see Fig. 12-36) and CDC7-DBF4. Both types of complexes help to activate replication by binding to and phosphorylating several subunits of the pre-RC. Other cyclins and CDKs function to inhibit the formation of more pre-RC complexes once replication has been initiated. For example, CDK2 binds to cyclin A as cyclin E levels decline during S phase, inhibiting CDK2 and preventing the licensing of additional pre-RC complexes.

The rate of movement of the replication fork in eukaryotes (∼50 nucleotides/s) is only one-twentieth that observed in E. coli. At this rate, replication of an average human chromosome proceeding from a single origin would take more than 500 hours, making the requirement for many origins evident.

Like bacteria, eukaryotes have several types of DNA polymerases. Some have been linked to particular functions, such as the replication of mitochondrial DNA. The replication of nuclear chromosomes primarily involves three multisubunit DNA polymerases. The highly processive DNA polymerase ε synthesizes the leading strand, and DNA polymerase $δ$ $bold-italic delta$ synthesizes the lagging strand. Both enzymes have $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ proofreading exonuclease activities. DNA polymerase α, a DNA polymerase/primase, synthesizes RNA primers and also extends them by about 10 nucleotides of DNA to initiate synthesis of each Okazaki fragment on the lagging strand. One subunit of DNA polymerase α has a primase activity, and the largest subunit $(M_{r} \sim 180, 000)$ $left-parenthesis upper M Subscript r Baseline tilde 180 comma 000 right-parenthesis$ contains the polymerization activity. However, this polymerase has no proofreading $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ exonuclease activity, making it unsuitable for high-fidelity DNA replication.

DNA polymerases ε and δ are associated with and stimulated by proliferating cell nuclear antigen (PCNA; $M_{r} 29, 000$ $upper M Subscript r Baseline 29 comma 000$ ), a protein found in large amounts in the nuclei of proliferating cells. The three-dimensional structure of PCNA is remarkably similar to that of the β subunit of E. coli DNA polymerase III (Fig. 25-8b), although primary sequence homology is not evident. PCNA has a function analogous to that of the β subunit, forming a circular clamp that enhances the processivity of the two polymerases.

Two other protein complexes also function in eukaryotic DNA replication. RPA (replication protein A) is a single-stranded DNA–binding protein, equivalent in function to the E. coli SSB protein. RFC (replication factor C) is a clamp loader for PCNA and facilitates the assembly of active replication complexes. The subunits of the RFC complex have significant sequence similarity to the subunits of the bacterial clamp-loading $(γ)$ $left-parenthesis gamma right-parenthesis$ complex.

Termination of replication on linear eukaryotic chromosomes occurs when replication forks operating from nearby origins converge. As in bacteria, there are successive steps of final replication, replisome dissociation, and decatenation of the DNA products. All of the steps are mediated by additional protein complexes, with some parts of the process still undefined.

Viral DNA Polymerases Provide Targets for Antiviral Therapy

Many DNA viruses encode their own DNA polymerases, and some of these have become targets for pharmaceuticals. For example, the DNA polymerase of the herpes simplex virus is inhibited by acyclovir, a compound developed by Gertrude Elion and George Hitchings (p. 836). Acyclovir consists of guanine attached to an incomplete ribose ring.

Acyclovir is a six-membered ring with N substituted for C at the bottom vertex, N substituted for C and bonded to H at the upper left vertex, a double bond at the lower left side, a lower left vertex bonded to N H 2, a top vertex double bonded to O, and a double bod at the right side shared with the left side of a five-membered ring with N substituted for C at its upper right vertex, a double bond at its upper right side, and N substituted for C at its lower right vertex that is further bonded to C H 2 bonded to O bonded to C H 2 C H 2 O H.

It is phosphorylated by a virally encoded thymidine kinase; acyclovir binds to this viral enzyme with an affinity 200-fold greater than its binding to the cellular thymidine kinase. This ensures that phosphorylation occurs mainly in virus-infected cells. Cellular kinases convert the resulting acyclo-GMP to acyclo-GTP, which is both an inhibitor and a substrate of DNA polymerases; acyclo-GTP competitively inhibits the herpes DNA polymerase more strongly than cellular DNA polymerases. Because it lacks a $3'$ $3 prime$ hydroxyl, acyclo-GTP also acts as a chain terminator when incorporated into DNA. Thus viral replication is inhibited at several steps.

SUMMARY 25.1 DNA Replication

Replication of DNA follows a set of universal rules. Replication is semiconservative, each strand acting as template for a new daughter strand. It is carried out in three identifiable phases: initiation, elongation, and termination. The process starts at a single origin in bacteria and usually proceeds bidirectionally. DNA is synthesized in the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ direction by DNA polymerases. At the replication fork, the leading strand is synthesized continuously in the same direction as replication fork movement; the lagging strand is synthesized discontinuously as Okazaki fragments, which are subsequently ligated.
Nucleases are enzymes that degrade DNA. Endonucleases cleave within a DNA polymer; exonucleases degrade DNA from the end of one strand (either $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ or $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ ).
DNA polymerases are complex enzymes that synthesize DNA, and often possess additional activities, including exonuclease functions.
DNA is replicated with very high fidelity. Accuracy is maintained by (1) base selection by the polymerase, (2) a $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ proofreading exonuclease activity that is part of many DNA polymerases, and (3) specific repair systems for mismatches left behind after replication.
Most cells have several DNA polymerases. In E. coli, DNA polymerase III is the primary replication enzyme. DNA polymerase I is responsible for special functions during replication, recombination, and repair.
Replication requires an array of enzymes and protein factors in addition to DNA polymerases. Many of these proteins belong to the $AAA + ATPase$ $AAA zero width space plus ATPase$ family.
Replication initiation occurs when replicative helicases are loaded onto replication origins in stepwise fashion. Elongation is achieved by an active replisome — a supramolecular complex of nucleic acids and many proteins, including polymerases. Termination occurs when replisomes proceeding in opposite directions converge. It requires decatenation of the replication products when replication is complete.
The major replicative DNA polymerases in eukaryotes are DNA polymerases ε and δ. DNA polymerase α synthesizes primers.
Viral DNA replication is a drug target.