25.2 DNA Repair in Chapter 25 DNA Metabolism

25.2 DNA Repair

Most cells have only one or two sets of genomic DNA. Damaged proteins and RNA molecules can be quickly replaced by using information encoded in the DNA, but DNA molecules themselves are irreplaceable. Maintaining the integrity of the information in DNA is a cellular imperative, supported by an elaborate set of DNA repair systems. DNA can become damaged by a variety of processes, some spontaneous, others catalyzed by environmental agents (Chapter 8). Replication itself can very occasionally damage the information content in DNA when polymerase errors create mismatched base pairs (such as G paired with T).

The chemistry of DNA damage is diverse and complex. The cellular response to this damage includes enzymatic systems that catalyze some of the most interesting chemical transformations in DNA metabolism. We first examine the effects of alterations in DNA sequence and then consider specific repair systems.

Mutations Are Linked to Cancer

The best way to illustrate the importance of DNA repair is to consider the effects of unrepaired DNA damage (a lesion). The most serious outcome is a change in the base sequence of the DNA, which, if replicated and transmitted to future generations of cells, becomes permanent. A permanent change in the nucleotide sequence of DNA is called a mutation. Mutations can involve the replacement of one base pair with another (substitution mutation) or the addition or deletion of one or more base pairs (insertion or deletion mutations). If the mutation affects nonessential DNA or if it has a negligible effect on the function of a gene, it is known as a silent mutation. Rarely, a mutation confers some biological advantage. Most nonsilent mutations, however, are neutral or deleterious.

In mammals there is a strong correlation between the accumulation of mutations and cancer. A simple test developed by Bruce Ames in the 1970s measures the potential of a given chemical compound to promote certain easily detected mutations in a specialized bacterial strain (Fig. 25-19). Few of the chemicals that we encounter in daily life score as mutagens in this test. However, of the compounds known to be carcinogenic from extensive animal trials, more than 90% are also found to be mutagenic in the Ames test. Because of this strong correlation between mutagenesis and carcinogenesis, the Ames test for bacterial mutagens is still widely used as a rapid and inexpensive screen for potential human carcinogens.

A four-part figure shows the Ames test for carcinogens by showing a strain of Salmonella typhimurium that cannot synthesize histidine growing in a medium without histidine in part a and growing with the same medium with discs containing different mutagens in parts b through d. — FIGURE 25-19 Ames test for carcinogens, based on their mutagenicity. A strain of *Salmonella typhimurium* having a mutation that inactivates an enzyme of the histidine biosynthetic pathway is plated on a histidine-free medium. Few cells grow. (a) The few small colonies of S. *typhimurium* that do grow on a histidine-free medium carry spontaneous mutations that permit the histidine biosynthetic pathway to operate. Three identical nutrient plates (b), (c), and (d) have been inoculated with an equal number of cells. Each plate then receives a disk of filter paper containing progressively lower concentrations of a mutagen. The mutagen greatly increases the rate of back-mutation and hence the number of colonies. The clear areas around the filter paper indicate where the concentration of mutagen is so high that it is lethal to the cells. As the mutagen diffuses away from the filter paper, it is diluted to sublethal concentrations that promote back-mutation. Mutagens can be compared on the basis of their effect on mutation rate. Because many compounds undergo a variety of chemical transformations after entering cells, compounds are sometimes tested for mutagenicity after first incubating them with a liver extract. Some substances have been found to be mutagenic only after this treatment. [Bruce N. Ames, University of California, Berkeley, Department of Biochemistry and Molecular Biology.]

The genomic DNA in a typical mammalian cell accumulates many thousands of lesions during a 24-hour period. However, as a result of DNA repair, fewer than 1 in 1,000 become a mutation. DNA is a relatively stable molecule, but in the absence of repair systems, the cumulative effect of many infrequent but damaging reactions would make life impossible.

All Cells Have Multiple DNA Repair Systems

The number and diversity of repair systems reflect both the importance of DNA repair to cell survival and the diverse sources of DNA damage (Table 25-5). Some common types of lesions, such as pyrimidine dimers (see Fig. 8-30), can be repaired by several distinct systems. Nearly 200 genes in the human genome encode proteins dedicated to DNA repair. In many cases, the loss of function of one of these proteins results in genomic instability and an increased occurrence of oncogenesis (Box 25-1).

TABLE 25-5 Types of DNA Repair Systems in *E. coli*

A table lists the types of D N A Repair Systems in E. coli.

The table consists of 2 columns and 20 rows. Column headings: Enzymes / proteins; Type of damage. Rows 1 through 10 are subcategorized as Mismatch repair. Row 1: D a m methylase; Mismatches. Row 2: M u t H, M u t L, M u t S proteins; Mismatches. Row 3: D N A helicase I I; Mismatches. Row 4: S S B; Mismatches. Row 5: D N A polymerase I II; Mismatches. Row 6: Exonuclease I; Mismatches. Row 7: Exonuclease V I I; Mismatches. Row 8: R e c J nuclease; Mismatches. Row 9: Exonuclease X; Mismatches. Row 10: D N A ligase; Mismatches. Rows 11 through 14 are subcategorized as Base-excision repair. Row 11: D N A glycosylases; Abnormal bases (uracil, hypoxanthine, xanthine), alkylated bases, in some other organisms, pyrimidine dimers. Row 12: A P endonuclease; Abnormal bases (uracil, hypoxanthine, xanthine), alkylated bases, in some other organisms, pyrimidine dimers. Row 13: D N A polymerase I; Abnormal bases (uracil, hypoxanthine, xanthine), alkylated bases, in some other organisms, pyrimidine dimers. Row 14: D N A ligase; Abnormal bases (uracil, hypoxanthine, xanthine), alkylated bases, in some other organisms, pyrimidine dimers. Rows 15 through 17 are subcategorized as Nucleotide-excision repair. Row 15: A B C excinuclease; D N A lesions that cause large structural change (e. g. pyrimidine dimers). Row 16: D N A polymerase I; D N A lesions that cause large structural change (e. g. pyrimidine dimers). Row 17: D N A ligase; D N A lesions that cause large structural change (e. g. pyrimidine dimers). Row 18 is subcategorized as Direct repair. Row 18: D N A photolyases; Pyrimidine dimers. Row 19: O superscript 6 methylguanine-D N A methyltransferase; O superscript 6 methylguanine. Row 20: A l k B protein; 1-Methylguanine, 3-methylcytosine.

BOX 25-1 MEDICINE

DNA Repair and Cancer

Human cancers develop when genes that regulate normal cell division (oncogenes and tumor suppressor genes; see Chapter 12) fail to function, are activated at the wrong time, or are altered. As a consequence, cells may grow out of control and form a tumor. The genes controlling cell division can be damaged by spontaneous mutation or overridden by the invasion of a tumor virus (Chapter 26). Not surprisingly, alterations in DNA repair genes that result in a higher rate of mutation can greatly increase an individual’s susceptibility to cancer. Defects in the genes encoding the proteins involved in nucleotide-excision repair, mismatch repair, recombinational repair, and error-prone translesion DNA synthesis have all been linked to human cancers. Clearly, DNA repair can be a matter of life and death.

Nucleotide-excision repair requires a larger number of proteins in humans than in bacteria, although the overall pathways are very similar. Genetic defects that inactivate nucleotide-excision repair have been associated with several genetic diseases, the best-studied of which is xeroderma pigmentosum (XP). Because nucleotide-excision repair is the sole repair pathway for pyrimidine dimers in humans, people with XP are extremely sensitive to light and readily develop sunlight-induced skin cancers. Most people with XP also have neurological abnormalities, presumably because of their inability to repair certain lesions caused by the high rate of oxidative metabolism in neurons. Defects in the genes encoding any of at least seven different protein components of the nucleotide-excision repair system can result in XP, giving rise to seven different genetic groups, denoted XPA to XPG. Note that XPC and XPE are parts of complexes that recognize damaged DNA, whereas XPA, XPB, XPD, XPF, and XPG are all components of a much larger multisubunit complex that represents the human excinuclease depicted in Figure 25-24. These proteins are involved in making the DNA incisions and removing the 29mer segment of DNA.

Most microorganisms have redundant pathways for the repair of cyclobutane pyrimidine dimers — making use of DNA photolyases and sometimes base-excision repair as alternatives to nucleotide-excision repair — but humans and other placental mammals do not. This lack of a backup for nucleotide-excision repair for removing pyrimidine dimers has led to speculation that early mammals were small, furry, nocturnal animals with little need to repair UV damage. However, mammals do have a pathway for the translesion bypass of cyclobutane pyrimidine dimers, which involves DNA polymerase $η$ $eta$ . This enzyme preferentially inserts two A residues opposite a T–T pyrimidine dimer, minimizing mutations. People with a genetic condition in which DNA polymerase $η$ $eta$ function is missing exhibit an XP-like illness known as XP-variant, or XP-V. Clinical manifestations of XP-V are similar to those of the classic XP diseases, although mutation levels are higher in XP-V when cells are exposed to UV light. Apparently, the nucleotide-excision repair system works in concert with DNA polymerase $η$ $eta$ in normal human cells, repairing and/or bypassing pyrimidine dimers as needed to keep cell growth and DNA replication going. Exposure to UV light introduces a heavy load of pyrimidine dimers, and some must be bypassed by translesion synthesis to keep replication on track. When one system is missing, it is partly compensated for by the other. A loss of DNA polymerase $η$ $eta$ activity leads to stalled replication forks and bypass of UV lesions by different, more mutagenic, translesion synthesis (TLS) polymerases. As when other DNA repair systems are absent, the resulting increase in mutations often leads to cancer.

One of the most common inherited cancer-susceptibility syndromes is hereditary nonpolyposis colon cancer (HNPCC). This syndrome has been traced to defects in mismatch repair. Human and other eukaryotic cells have several proteins analogous to the bacterial MutL and MutS proteins (see Fig. 25-21). Defects in at least five different mismatch repair genes can give rise to HNPCC. The most prevalent are defects in the hMLH1 (human MutL homolog 1) and hMSH2 (human MutS homolog 2) genes. In individuals with HNPCC, cancer generally develops at an early age, with colon cancers being most common.

Most human breast and ovarian cancer occurs in women with no known predisposition. However, about 10% of cases are associated with inherited defects in two genes, BRCA1 and BRCA2. Human BRCA1 and BRCA2 are large proteins (1,834 and 3,418 amino acid residues, respectively) that interact with a wide range of other proteins involved in transcription, chromosome maintenance, DNA repair, and control of the cell cycle. BRCA2 has been implicated in the recombinational DNA repair of double-strand breaks. One of the key roles of BRCA2 is to load the human RecA homolog, called Rad51, onto DNA at the sites of double-strand breaks. BRCA1 has as yet imperfectly defined roles in the repair of double-strand breaks, transcription, and some other processes of DNA metabolism. Women with defects in either the BRCA1 or BRCA2 gene have a high (~70%) chance of developing breast cancer.

Many DNA repair processes also seem to be extraordinarily inefficient energetically — an exception to the pattern observed in the vast majority of metabolic pathways, where every ATP is generally accounted for and used optimally. When the integrity of the genetic information is at stake, the amount of chemical energy invested in a repair process seems almost irrelevant.

Accurate DNA repair is possible largely because the DNA molecule consists of two complementary strands. Damaged DNA in one strand can be removed and replaced, without introducing mutations, by using the undamaged complementary strand as a template. We consider here the principal types of repair systems, beginning with those that repair the rare nucleotide mismatches that are left behind by replication.

Mismatch Repair

Correction of the rare mismatches left after replication in E. coli improves the overall fidelity of replication by an additional factor of $10^{2} to 10^{3}$ $10 squared to 10 cubed$ . The mismatches are nearly always corrected to reflect the information in the old (template) strand, which the repair system can distinguish from the newly synthesized strand by the presence of methyl group tags on the template DNA. The mismatch repair system of E. coli includes at least 10 protein components (Table 25-5) that function either in strand discrimination or in the repair process itself. The functions of many of these were first worked out by Paul Modrich and colleagues in the 1980s.

The strand discrimination mechanism has not been determined for most bacteria or eukaryotes, but it is well understood for E. coli and some closely related bacterial species. In these bacteria, strand discrimination is based on the action of Dam methylase, which, as you will recall, methylates DNA at the $N^{6}$ $upper N Superscript 6$ position of all adenines within $(5')$ $left-parenthesis 5 prime right-parenthesis$ GATC sequences. Immediately after passage of the replication fork, there is a short period (a few seconds or minutes) during which the template strand is methylated but the newly synthesized strand is not (Fig. 25-20). The transient unmethylated state of GATC sequences in the newly synthesized strand permits the new strand to be distinguished from the template strand. Replication mismatches in the vicinity of a hemimethylated GATC sequence are then repaired according to the information in the methylated parent (template) strand. If both strands are methylated at a GATC sequence, few mismatches are repaired; if neither strand is methylated, repair occurs but does not favor either strand. The methyl-directed mismatch repair system of E. coli efficiently repairs mismatches up to 1,000 bp from a hemimethylated GATC sequence.

A figure shows how methylation can be used to distinguish strands, which is important in mismatch repair. — FIGURE 25-20 Methylation and mismatch repair. Methylation of DNA strands can serve to distinguish parent (template) strands from newly synthesized strands in *E. coli* DNA, a function that is critical to mismatch repair. The methylation occurs at the $N^{6}$ $upper N Superscript 6$ of adenines in $(5') GATC$ $left-parenthesis 5 prime right-parenthesis GATC$ sequences. This sequence is a palindrome, present in opposite orientations on the two strands.

FIGURE 25-20 Methylation and mismatch repair. Methylation of DNA strands can serve to distinguish parent (template) strands from newly synthesized strands in *E. coli* DNA, a function that is critical to mismatch repair. The methylation occurs at the $N^{6}$ $upper N Superscript 6$ of adenines in $(5') GATC$ $left-parenthesis 5 prime right-parenthesis GATC$ sequences. This sequence is a palindrome, present in opposite orientations on the two strands.

A double-stranded piece of D N A consists of two horizontal parallel blue lines with vertical lines extending down from the top strand and up from the bottom strand to indicate base pairing. The top strand runs from 5 prime to 3 prime and the bottom strand runs from 3 prime to 5 prime. Bases three through six are labeled in both strands. In the top strand, these are C, A bonded to red highlighted C H 3 above, T and C. In the bottom strand, these are C, T, A bonded to red highlighted C H 3 below, and G. An arrow points down labeled replication. This yields a molecule in which the right side is the same but there is a replication fork that separates two halves on the left side. A red arrow points from left to right into the fork. The top strand is still blue, still runs from 5 prime to 3 prime, and has the same bases labeled. The bottom strand is tan, begins at the 3 prime end on the left, and matches the blue strand above. The bases C, T, A and G are shown in positions 3 through 6 beneath the labeled bases in the blue strand. The bottom half is similar except that the bottom strand is still blue, still runs from 3 prime to 5 prime, and still has the same bases labeled. Its complementary strand is tan, begins at the 5 prime end, and has the bases G, A, T, and C shown in positions 3 through 6 above the labeled bases in the bottom strand. An arrow points down accompanied by a gray box reading, For a short period following replication, the template strand is methylated and the new strand is now. Two double-stranded pieces of D N A are shown with text reading, hemimethylated D N A between them. The top D N A molecule has a top blue strand that runs from 5 prime to 3 prime with bases 3 through 6 labeled a G, A bonded to red highlighted C H 3, T and C. The bottom strand is tan and has bases 3 through 6 labeled as C, T, A and G. The bottom D N A molecule is similar except that the top strand is tan and the bottom strand is blue, A in position four on the top strand is not methylated, and A in position five on the bottom strand is bonded to red highlighted C H 3. An arrow labeled blue highlighted dam methylase is accompanied by a gray box reading, After a few minutes, the new strand is methylated and the two strands can no longer be distinguished. Two double-stranded molecules of D N A are shown that both have the same structure as at the beginning. Each has A in position 4 on the top strand bonded to red highlighted C H 3 and A in position 5 on the bottom strand bonded to red highlighted C H 3.

How is the mismatch correction process directed by relatively distant GATC sequences? Figure 25-21 illustrates one mechanism. MutS scans the DNA and forms a clamplike complex upon encountering a lesion. The complex binds to all mismatched base pairs (except C–C). MutL protein forms a complex with MutS protein, and the MutSL complex slides along the DNA to find a hemimethylated GATC sequence. MutH binds to MutL, and the MutSLH complex moves in either direction at random along the DNA. MutH has a site-specific endonuclease activity that is inactive until the complex encounters a hemimethylated GATC sequence. At this site, MutH catalyzes cleavage of the unmethylated strand on the $5'$ $5 prime$ side of the G in GATC, which marks the strand for repair. Further steps in the pathway depend on where the mismatch is located relative to this cleavage site (Fig. 25-22).

A figure shows a model for the early steps of methyl-directed mismatch repair. — FIGURE 25-21 A model for the early steps of methyl-directed mismatch repair. Recognition of the sequence $(5') GATC$ $left-parenthesis 5 prime right-parenthesis GATC$ and of the mismatch are specialized functions of the MutH and MutS proteins, respectively.

FIGURE 25-21 A model for the early steps of methyl-directed mismatch repair. Recognition of the sequence $(5') GATC$ $left-parenthesis 5 prime right-parenthesis GATC$ and of the mismatch are specialized functions of the MutH and MutS proteins, respectively.

A double-stranded piece of D N A is shown with a blue top strand that runs from 5 prime to 3 prime and a bottom tan strand that runs from 3 prime to 5 prime. The top blue strand has a bond to red highlighted C H 3 near its left end. Above halfway across the molecule, the blue strand has a sharp point upward (like a triangle with no base) and the tan strand has a similar sharp point downward. This is labeled, mismatched base pair. A close-up of a piece of D N A to the right shows a top blue strand with eight short vertical lines pointing down to represent bases and a bottom tan strand with eight short vertical lines pointing up to represent bases. Positions three through six are labeled on both strands. On the top strand, positions three through six contain G, A bonded to red highlighted C H 3, T, and C. On the bottom strand, positions three through six contain C, T, A, and G. An arrow points down accompanied by a curved arrow showing the addition of an orange oval labeled A T P and loss of A D P plus P subscript i end subscript. A curved line shows the addition of blue highlighted M u t S as A T P is added and a second curved line shows the addition of blue highlighted M u t L after A D P plus P I leaves. M u t S is shown as a purple oval with an oval opening near the bottom and M u t L is shown as a roughly rectangular structure with a round opening inside that extends to a narrow opening at the bottom. This yields a double-stranded piece of D N A similar to the top strand except that the two strands run through the opening in M u t S just before the mismatch and through the opening in M u t L just after the mismatch. The opening at the bottom of M u t L is closed in this illustration. This structure is labeled M u t S L complex. To the right, a teal circle labeled M u t H overlaps both strands just to the left of a bond from the blue strand to red highlighted C H 3. An arrow points down accompanied by a curved arrow showing the addition of an orange oval labeled A T P and loss of A D P plus P subscript i end subscript. Blue highlighted M u t H is added at the same time as A T P. Text in a gray box to the right reads, M u t S L complex forms around the D N A at the mismatch and moves along the D N A to M u t H bound at a hemimethylated G A T C. A similar D N A molecule is shown with the mismatch to the left of the M u t S L complex. This complex now shows a purple oval with two openings, with the blue strand passing behind the left side of the top opening and out across the right side of the top opening across the upper left of M u t L to disappear behind the right side of M u t L. A teal sphere of M u t H overlaps the right side of M u t L just before a bond from the blue top strand to red highlighted C H 3 above. An arrow points down accompanied by text in a gray box reading, M u t H cleaves the unmethylated strand on the 5 prime side of G in the G A T C sequence. This yields a similar strand of D N A with a blue top strand and tan bottom strand and with a mismatched base pair in the middle. There is a cut in the tan strand just beneath the bond from the blue strand to red highlighted C H 3 above on the right.

A figure shows the completion of methyl-directed mismatch repair. — FIGURE 25-22 Completion of methyl-directed mismatch repair. The combined action of DNA helicase II, SSB, and one of four different exonucleases removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch. The particular exonuclease depends on the location of the cleavage site relative to the mismatch, as shown by the alternative pathways here. The resulting gap is filled in (dashed line) by DNA polymerase III, and the nick is sealed by DNA ligase (not shown).

A double-stranded piece of D N A is shown with a blue top strand that runs from 3 prime to 5 prime and a tan bottom strand that runs from 5 prime to 3 prime. A mismatched base pair is shown halfway across the molecule as a sharp upward point in the blue strand above a sharp downward point in the tan strand. The blue strand has bonds to red highlighted C H 3 on the left and right sides. An arrow points down accompanied by a curved arrow showing the addition of an orange oval labeled A T P and loss of A D P plus P subscript i end subscript and curved lines show the addition of blue highlighted M u t S, M u t L, and M u t H. The arrow branches to the left and right. The left- and right-hand arrows both point to similar structures below, but the left-hand structure has an opening in the tan strand beneath the left-hand C H 3 and the right-hand structure has an opening in the tan strand beneath the right-hand C H 3. On both sides, arrows point down. On the right, the downward arrow is accompanied by curved arrows showing the addition of an orange oval labeled A T P and loss of A D P plus P subscript i end subscript and by curved lines showing the addition of blue highlighted M u t S L, D N A helicase Roman numeral 2, S S B, exonuclease Roman numeral 7, or Rec J nuclease. This yields a similar molecule in which there is a gap in the tan strand between the position beneath red highlighted C H 3 on the left and the downward point representing the mismatch. An arrow points down accompanied by text reading blue highlighted D N A polymerase Roman numeral 3, S S B, D N A ligase. This yields a similar molecule with a dashed tan arrow pointing from the end of the existing tan line just below the bond from the blue strand to red highlighted C H 3 above and the position where the mismatch had occurred. The point in the blue strand is no longer present, showing that there is no longer a mismatch. The right-hand series of reactions that occur after the initial cut beneath the bond to C H 3 on the right is similar. A downward arrow is accompanied by curved arrows showing the addition of an orange oval labeled A T P and loss of A D P plus P subscript i end subscript and by curved lines showing the addition of blue highlighted M u t S L, D N A helicase Roman numeral 2, S S B, exonuclease Roman numeral I or exonuclease Roman numeral 7 or exonuclease Roman numeral 10. This yields a similar molecule in which there is a gap in the tan strand between the position beneath red highlighted C H 3 on the right and the downward point representing the mismatch. An arrow points down accompanied by text reading blue highlighted D N A polymerase Roman numeral 3, S S B, D N A ligase. This yields a similar molecule with a dashed tan arrow pointing from the former location of the mismatch to the beginning of the existing tan line just past the bond from the blue strand to red highlighted C H 3 above. The point in the blue strand is no longer present, showing that there is no longer a mismatch.

When the mismatch is on the $5'$ $5 prime$ side of the cleavage site (Fig. 25-22, right side), the unmethylated strand is unwound and degraded in the $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ direction from the cleavage site through the mismatch, and this segment is replaced with new DNA. This process requires the combined action of DNA helicase II (also called UvrD helicase), SSB, exonuclease I or exonuclease X (both of which degrade strands of DNA in the $3^{'} \to 5^{'}$ $3 prime right-arrow 5 prime$ direction) or exonuclease VII (which degrades single-stranded DNA in either direction), DNA polymerase III, and DNA ligase. The pathway for repair of mismatches on the $3'$ $3 prime$ side of the cleavage site is similar (Fig. 25-22, left), except that the exonuclease is either exonuclease VII or RecJ nuclease (which degrades single-stranded DNA in the $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ direction).

Mismatch repair is particularly costly for E. coli in terms of energy expended. The mismatch may occur 1,000 or more base pairs from the GATC sequence. The degradation and replacement of a strand segment of this length require an enormous investment in activated deoxynucleotide precursors to repair a single mismatched base. This again underscores the importance to the cell of genomic integrity.

Eukaryotic cells also have mismatch repair systems, with several proteins structurally and functionally analogous to the bacterial MutS and MutL (but not MutH) proteins. Alterations in human genes encoding proteins of this type produce some of the most common inherited cancer-susceptibility syndromes (see Box 25-1), further demonstrating the value to the organism of DNA repair systems. The main MutS homologs in most eukaryotes, from yeast to humans, are MSH2 (MutS homolog), MSH3, and MSH6. Heterodimers of MSH2 and MSH6 generally bind to single base-pair mismatches, and they bind less well to slightly longer mispaired loops. In many organisms, the longer mismatches (2 to 6 bp) may be bound instead by a heterodimer of MSH2 and MSH3, or are bound by both types of heterodimers in tandem. Homologs of MutL, predominantly a heterodimer of MLH1 (MutL homolog) and PMS1 (post-meiotic segregation), bind to and stabilize the MSH complexes. Many details of the subsequent events in eukaryotic mismatch repair remain to be worked out. In particular, we do not know how newly synthesized DNA strands are identified, although research reveals that this process does not involve GATC sequences.

Base-Excision Repair

Every cell has a class of enzymes called DNA glycosylases that recognize particularly common DNA lesions (such as the products of cytosine and adenine deamination; see Fig. 8-29a) and remove the affected base by cleaving the N-glycosyl bond. The repair pathway is called base-excision repair, as the first step involves only the removal of the base rather than an entire nucleotide. The cleavage creates an apurinic or apyrimidinic site in the DNA, commonly referred to as an AP site or abasic site. Each DNA glycosylase is generally specific for one type of lesion.

Uracil DNA glycosylases, for example, found in most cells, specifically remove from DNA the uracil that results from spontaneous deamination of cytosine. Mutant cells that lack this enzyme have a high rate of $G \equiv C$ $upper G identical-to upper C$ to $A ═ T$ $upper A box drawings double horizontal upper T$ mutations. This glycosylase does not remove uracil residues from RNA or thymine residues from DNA. The capacity to distinguish thymine from uracil, the product of cytosine deamination — necessary for the selective repair of the latter — may be one reason why DNA evolved to contain thymine instead of uracil (p. 280).

Most bacteria have just one type of uracil DNA glycosylase, whereas humans have at least four types, with different specificities — an indicator of the importance of removing uracil from DNA. The most abundant human uracil glycosylase, UNG, is associated with the replisome, where it eliminates the occasional U residue inserted in place of a T during replication. The deamination of C residues is 100-fold faster in single-stranded DNA than in double-stranded DNA, and humans have an enzyme, hSMUG1, that removes any U residues occurring in single-stranded DNA during replication or transcription. Two other human DNA glycosylases, TDG and MBD4, remove either U or T residues paired with G, which are generated by deamination of cytosine or 5-methylcytosine, respectively.

Other DNA glycosylases recognize and remove a variety of damaged bases, including formamidopyrimidine and 8-hydroxyguanine (both arising from purine oxidation), hypoxanthine (from adenine deamination), and alkylated bases such as 3-methyladenine and 7-methylguanine. Glycosylases that recognize other lesions, including pyrimidine dimers, have also been identified in some classes of organisms. Remember that AP sites also arise from slow, spontaneous hydrolysis of the N-glycosyl bonds in DNA (see Fig. 8-29b).

Once an AP site has been formed by a DNA glycosylase, another type of enzyme must repair it. The repair is not made by simply inserting a new base and re-forming the N-glycosyl bond. Instead, the deoxyribose $5'$ $5 prime$ -phosphate left behind is removed and replaced with a new nucleotide. This process begins with one of the AP endonucleases, enzymes that cut the DNA strand containing the AP site. The position of the incision relative to the AP site ( $5'$ $5 prime$ or $3'$ $3 prime$ to the site) depends on the type of AP endonuclease. A segment of DNA including the AP site is then removed, DNA polymerase I replaces the DNA, and DNA ligase seals the remaining nick (Fig. 25-23). In eukaryotes, nucleotide replacement is carried out by specialized polymerases, as described below.

A figure shows the base-excision repair pathway for D N A repair. — FIGURE 25-23 DNA repair by the base-excision repair pathway. A DNA glycosylase recognizes a damaged base (in this case, a uracil) and cleaves between the base and deoxyribose in the backbone. An AP endonuclease cleaves the phosphodiester backbone near the AP site. DNA polymerase I initiates repair synthesis from the free $3'$ $3 prime$ hydroxyl at the nick, removing (with its $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease activity) and replacing a portion of the damaged strand. The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.

FIGURE 25-23 DNA repair by the base-excision repair pathway. A DNA glycosylase recognizes a damaged base (in this case, a uracil) and cleaves between the base and deoxyribose in the backbone. An AP endonuclease cleaves the phosphodiester backbone near the AP site. DNA polymerase I initiates repair synthesis from the free $3'$ $3 prime$ hydroxyl at the nick, removing (with its $5^{'} \to 3^{'}$ $5 prime right-arrow 3 prime$ exonuclease activity) and replacing a portion of the damaged strand. The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.

Two horizontal strands of D N A are shown. The top strand runs from 5 prime to 3 prime and the bottom strand runs from 3 prime to 5 prime. Both strands begin with a blue sugar on the left bonded to a blue circle labeled P bonded to a sugar further bonded to P and so on. The sequence of bases on the top strand is G, A, C, U, G, A, C, A, and C. The sequence of bases on the bottom strand is C, T, G, G, C, T, G, T G. All of the bases are properly paired except for U, which is shown in brown with two hydrogen bonds to G below. Step 1: An arrow points down accompanied by blue highlighted text reading D N A glycosylase (uracil glycosylase) and is accompanied by a branched arrow showing the loss of a brown hexagonal U. This yields a similar molecule in which there is a gap where U had been. Step 2: An arrow points down accompanied by blue text reading, A P endonuclease. This yields a similar molecule in which the bond between sugar bound to C to the left of where U had been is no longer bonded to P bonded to the sugar that had previously been bonded to U. This creates a break in the molecule and moves the sugar and phosphate associated with U upward. Step 3: An arrow labeled blue highlighted D N A polymerase Roman numeral 1 is accompanied by a curved arrow showing the addition of N T P s and loss of deoxyribose phosphate plus d N M Ps. This yields a similar piece of D N A in which the uracil has been replaced by a C at the fourth position from the left and the nucleotides in the further through eighth positions from the left have all been replaced by new D N A indicated by red sugars and red circles around P. There is a nick between the eighth sugar and the blue P to its right, creating an opening in the strand. Step 4: An arrow points down accompanied by blue highlighted text reading, D N A ligase. The nick is sealed and the red sugar at the eighth position from the left is now bonded to the blue circle labeled P bonded to the blue sugar at the ninth position from the left.

Nucleotide-Excision Repair

DNA lesions that cause large distortions in the helical structure of DNA generally are repaired by the nucleotide-excision system, a repair pathway critical to the survival of all free-living organisms. In nucleotide-excision repair (Fig. 25-24), a multisubunit enzyme (excinuclease) hydrolyzes two phosphodiester bonds, one on either side of the distortion caused by the lesion. In E. coli and other bacteria, the enzyme system hydrolyzes the fifth phosphodiester bond on the $3'$ $3 prime$ side and the eighth phosphodiester bond on the $5'$ $5 prime$ side to generate a fragment of 12 to 13 nucleotides (depending on whether the lesion involves one or two bases). In humans and other eukaryotes, the enzyme system hydrolyzes the sixth phosphodiester bond on the $3'$ $3 prime$ side and the twenty-second phosphodiester bond on the $5'$ $5 prime$ side, producing a fragment of 27 to 29 nucleotides. Following the dual incision, the excised oligonucleotides are released from the duplex and the resulting gap is filled — by DNA polymerase I in E. coli and DNA polymerase ε in humans. DNA ligase seals the nick.

FIGURE 25-24 Nucleotide-excision repair in *E. coli* and humans. The general pathway of nucleotide-excision repair is similar in all organisms. An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on either side of the lesion. The DNA segment — of 13 nucleotides (13mer) or 29 nucleotides (29mer) — is removed with the aid of a helicase. The gap is filled in by DNA polymerase, and the remaining nick is sealed with DNA ligase. [Information from a figure provided by Aziz Sancar.]

At the top of the illustration, a double stranded D N A molecule has two blue strands. The top strand runs from 5 prime to 3 prime with vertical lines extending down to represent bases and the bottom strand runs from 3 prime to 5 prime with vertical lines extending up to represent bases. At about three-quarters of the way across the molecule, after 14 blue bases, there is a red portion in the top strand where the strand bends up diagonally, runs horizontally, and then bends down diagonally again to continue with five blue vertical bases. There is a red box beneath the horizontal piece and one red line extends from each diagonal piece. This red region is labeled D N A lesion. An arrow points downward and splits to show italicized E coli end italics on the left and human on the right. Step 1: An arrow labeled blue highlighted excinuclease points downward on the left and on the right. For the bacterium, this yields a short blue piece of D N A with eight bases, then a gap, then the ninth base bonded to a circle labeled P to the upper left, then additional bases and the lesion, then three blue bases after the lesion before another gap followed by a base bonded to a circle labeled P. For the human, the process is similar but the piece of D N A is mch longer. There is a space after the fifth base from the left, a sixth base bonded to a circle labeled P, and then the lesion begins after the twenty sixth blue base. There are four base after the lesion, then a gap, then a blue base bonded to a circle labeled P followed by a final blue base. Step 2: An arrow labeled blue highlighted D N A helicase points downward. For the bacterium, a branched arrow shows the loss of a piece of blue D N A containing the lesion with text above the lesion reading, 13 mer. There are thirteen bases in total in the piece that is removed. This results in a strand of D N A in which the eighth blue base is now bonded to O H, then there is a gap of thirteen bases, then there is a blue base bonded to a circle labeled P followed by a final blue base. For the human, a branched arrow shows the loss of a long piece containing a lesion labeled 29 mer. There are 29 bases total in the piece that is removed. This leaves a long piece of single stranded D N A. On the left, there are five bases on the top strand with the fifth base bonded to O H. On the right, the second to last base is bonded to a circle labeled P and then the strand ends with a final blue base. Step 3: Arrows point down on each side. The arrow for italicized E. coli end italics is labeled blue highlighted D N A polymerase Roman numeral 1 and this yields a similar molecule in which the missing blue nucleotides are replaced by red nucleotides with a gap remaining between the right-hand red nucleotide and the blue nucleotide on the right bonded to a circle labeled P. The arrow for humans is labeled blue highlighted D N A polymerase epsilon and also yields a similar molecule in which the missing blue nucleotides are replaced by red nucleotides with a gap remaining between the right-hand red nucleotide and the blue nucleotide on the right bonded to a circle labeled P. Step 4: Arrows labeled blue highlighted D N A ligase. In both cases, a similar product is formed but the gap has been removed and no bases are bonded to circles labeled P.

In E. coli, the key enzymatic complex is the ABC excinuclease, which has three protein components, UvrA $(M_{r} 104, 000)$ $left-parenthesis upper M Subscript r Baseline 104 comma 000 right-parenthesis$ , UvrB $(M_{r} 78, 000)$ $left-parenthesis upper M Subscript r Baseline 78 comma 000 right-parenthesis$ , and UvrC $(M_{r} 68, 000)$ $left-parenthesis upper M Subscript r Baseline 68 comma 000 right-parenthesis$ . The term “excinuclease” is used to describe the unique capacity of this enzyme complex to catalyze two specific endonucleolytic cleavages, distinguishing this activity from that of standard endonucleases. A dimeric UvrA protein (an ATPase) scans the DNA and binds to the site of a lesion. A UvrB protein can bind to UvrA either before or after an encounter with the lesion. At the lesion, the UvrA dimer dissociates, leaving a tight UvrB-DNA complex. UvrC protein then binds to UvrB, and UvrB makes an incision at the fifth phosphodiester bond on the $3'$ $3 prime$ side of the lesion. This is followed by a UvrC-mediated incision at the eighth phosphodiester bond on the $5'$ $5 prime$ side. The resulting fragment, consisting of 12 to 13 nucleotides, is removed by UvrD helicase. The short gap thus created is filled in by DNA polymerase I and DNA ligase. This pathway (Fig. 25-24, left) is a primary repair route for many types of lesions, including cyclobutane pyrimidine dimers, 6-4 photoproducts (see Fig. 8-30), and several other types of base adducts, including benzo $[a]$ $left-bracket a right-bracket$ pyrene-guanine, which is formed in DNA by exposure to cigarette smoke. The nucleolytic activity of the ABC excinuclease is novel in the sense that two cuts are made in the DNA.

The mechanism of eukaryotic excinucleases is quite similar to that of the bacterial enzyme, although at least 16 polypeptides with no similarity to the E. coli excinuclease subunits are required for the dual incision. Some of the nucleotide-excision repair and base-excision repair in eukaryotes is closely tied to transcription (see Chapter 26). Genetic deficiencies in nucleotide-excision repair in humans give rise to a variety of serious diseases (see Box 25-1).

Direct Repair

Several types of damage are repaired without removing a base or nucleotide. The best-characterized example is direct photoreactivation of cyclobutane pyrimidine dimers, a reaction promoted by DNA photolyases. Pyrimidine dimers result from a UV-induced reaction. Through a mechanism worked out by Aziz Sancar and colleagues, photolyases use energy derived from absorbed light to reverse the damage (Fig. 25-25). Photolyases generally contain two cofactors that serve as light-absorbing agents, or chromophores: in all organisms, one is ${FADH}_{2}$ $FADH Subscript 2$ ; in E. coli and yeast, the other is a folate. The reaction mechanism entails the generation of free radicals. DNA photolyases are not found in humans and other placental mammals.

A figure shows the repair of pyridine dimers with photolyase. — MECHANISM FIGURE 25-25 Repair of pyrimidine dimers with photolyase. Energy derived from absorbed light is used to reverse the photoreaction that caused the lesion. The two chromophores in *E. coli* photolyase $(M_{r} 54, 000)$ $left-parenthesis upper M Subscript r Baseline 54 comma 000 right-parenthesis$ , $N^{5}, N^{10} -methenyltetrahydrofolylpolyglutamate$ $upper N Superscript 5 Baseline comma upper N Superscript 10 Baseline hyphen methenyltetrahydrofolylpolyglutamate$ (MTHFpolyGlu) and ${FADH}^{-}$ $FADH Superscript minus$ , perform complementary functions. MTHFpolyGlu functions as a photoantenna to absorb blue-light photons. The excitation energy passes to ${FADH}^{-}$ $FADH Superscript minus$ , and the excited flavin $(* {FADH}^{-})$ $left-parenthesis asterisk FADH Superscript minus Baseline right-parenthesis$ donates an electron to the pyrimidine dimer, leading to the rearrangement as shown.

MECHANISM FIGURE 25-25 Repair of pyrimidine dimers with photolyase. Energy derived from absorbed light is used to reverse the photoreaction that caused the lesion. The two chromophores in *E. coli* photolyase $(M_{r} 54, 000)$ $left-parenthesis upper M Subscript r Baseline 54 comma 000 right-parenthesis$ , $N^{5}, N^{10} -methenyltetrahydrofolylpolyglutamate$ $upper N Superscript 5 Baseline comma upper N Superscript 10 Baseline hyphen methenyltetrahydrofolylpolyglutamate$ (MTHFpolyGlu) and ${FADH}^{-}$ $FADH Superscript minus$ , perform complementary functions. MTHFpolyGlu functions as a photoantenna to absorb blue-light photons. The excitation energy passes to ${FADH}^{-}$ $FADH Superscript minus$ , and the excited flavin $(* {FADH}^{-})$ $left-parenthesis asterisk FADH Superscript minus Baseline right-parenthesis$ donates an electron to the pyrimidine dimer, leading to the rearrangement as shown.

M T H F poly G l u is shown at the upper left as a left-hand six-membered ring with N substituted for C at the bottom vertex and bonded to H, N substituted for C at the upper left vertex and bonded to H, the lower left vertex bonded to N H 2, the top vertex double bonded to O, and a double bond at the right side shared with the left side of an adjacent central six-membered ring with N substituted for C at the bottom vertex and bonded to H, N plus substituted for C at the top vertex and double bonded to C H bonded to N that is further bonded to the left side vertex of a six-membered ring and to C H 2 tot eh lower left further bonded to the upper right vertex of the central six-membered ring. The right-hand six-membered ring has double bonds at it slower left and lower right sides and a right side vertex bonded to (C l u) subscript italicized n end italics end subscript. Step 1: A wavy yellow to red arrow labeled light points to a right-pointing arrow above text in a yellow box reading, A blue-light photon (300 to 500 n m) is absorbed by M T H F poly G l u. This yields asterisk M T H F poly G l u, which is shown within brackets as a similar molecule. Step 2: A dashed blue arrow points from asterisk M T H F poly G l u to an arrow in a cycle pointing from F A D H minus to asterisk F A D H minus. A yellow box contains accompanying text reading, The excitation energy passes to F A D H minus in the active site. A cycle of reactions is shown partly in a brown box. F A D H minus on the right side within the box is a benzene ring with its upper and lower left vertices bonded to C H 3 and a double bond at its right side shared with the left side of a central six-membered ring with N substituted for C at its bottom vertex and bonded to H, N substituted for C at its top vertex and bonded to R, and a double bond at its right side shared with the left side of a right-hand six-membered ring. This right-hand six-membered ring has N substituted for C at its lower right vertex and bonded to H, a bottom vertex double bonded to O, an upper right vertex double bonded to O, and N minus substituted for C at its top vertex. An arrow points from F A D H minus to asterisk F A D H minus at the top center of the cycle. Asterisk F A D H minus is shown in brackets and resembles F A D H minus. An arrow from asterisk F A D H minus to flavin radical F A D H dot below meets a downward arrow labeled 3 next to e minus outside of the brown box. The flavin radical resembles asterisk F A D H minus except that N substituted for C at the bottom vertex of the central ring and bonded to H has a red dot and a positive charge. An arrow points right from the flavin radical F A D H dot and is joined by an arrow showing the addition of e minus from step 5 below to form F A D H minus to continue to cycle in the brown box. Step 3 is a downward-pointing arrow /labeled by a yellow box that reads, The excited flavin (asterisk F A D H minus) donates an electron to the pyrimidine dimer to generate an unstable dimer radical. The reactant is a cyclobutene pyrimidine dimer. This has a square ring that shares its right side with the left side of a right-hand six-membered ring and its left side with the right side of a left-hand six-membered ring. The right-hand six-membered ring has N substituted for C at the upper right vertex and bonded to H, a top vertex double bonded to O, a lower right vertex double bonded to O, and N substituted for C at the bottom vertex that is further bonded below to C H bonded to C H 3 to the right and to C H 2 below bonded to a circle labeled P to the left further bonded to C H bonded to C H 2 C H 3 below and to N substituted for C at the bottom vertex of the left-hand six-membered ring. The left-hand six-membered ring also has N substitute for C at its upper left vertex and bonded to H and its lower left and top vertices double bonded to O. The arrow labeled step 3 points down accompanied by e minus from the conversion of asterisk F A D H minus to flavin radical F A D H dot to yield a similar molecule in which the left-hand six-membered ring has a top vertex with a single red dot and a bond to O minus above. Red fishhook arrows point from the red dot to the upper right side bond and from the top bond of the central square ring to the upper right bond of the left-hand ring and the upper let bond of the right-hand ring. Step 4 is an arrow pointing down from this molecule to a similar product. The product is similar except there is no top bond connecting the upper left and right vertices of the six-membered rings to form a square ring and there is a red dot at the upper left vertex of the right-hand six-membered ring. Red fishhook arrows point from the red dot to the left side of the right-hand six-membered ring and from the bond connecting the lower right vertex of the left-hand ring and the lower left vertex of the right-hand ring, which had previously been the bottom of the square ring, to the lower right vertex of the left-hand ring and the left side of the right-hand ring. Another arrow labeled 4 points to a product with two six-membered rings with no square ring between them. Step 4 is accompanied by a yellow box containing text reading, electron rearrangement restores monomeric pyrimidines. The left-hand ring has the same structure as before except that there is a red dot at the lower right vertex instead of a bond. The right-hand ring has a double bond at its left side. Red fishhook arrows point from the red dot and from the double bond at the upper right side to the right side of the left-hand ring. A red arrow points from O minus to the bond between O minus and the top vertex of the left-hand ring. Step 5: An arrow points right to show that this yields monomeric pyrimidines in repaired D N A and an arrow branches away from this arrow showing that e minus joins with an arrow from flavin radical F A D H dot to form F A D H minus. The monomeric pyrimidines in repaired D N A are similar to the reactants except that the left-hand ring has a double bond at its right side and a double bond from it stop vertex to O.

Additional examples are seen in the repair of nucleotides with alkylation damage. The modified nucleotide $O^{6}$ $upper O Superscript 6$ -methylguanine forms in the presence of alkylating agents and is a common and highly mutagenic lesion. It tends to pair with thymine rather than cytosine during replication, and therefore causes $G \equiv C$ $upper G identical-to upper C$ to $A ═ T$ $upper A box drawings double horizontal upper T$ mutations (Fig. 25-26). Direct repair of $O^{6}$ $upper O Superscript 6$ -methylguanine is carried out by $O^{6}$ $upper O Superscript 6$ -methylguanine-DNA methyltransferase, a protein that catalyzes transfer of the methyl group of $O^{6}$ $upper O Superscript 6$ -methylguanine to one of its own Cys residues. This methyltransferase is not strictly an enzyme, because a single methyl transfer event permanently methylates the protein, inactivating it in this pathway. The consumption of an entire protein molecule to correct a single damaged base is another vivid illustration of the priority given to maintaining the integrity of cellular DNA.

A figure shows the conversion of an O superscript 6 end superscript-methylguanine nucleotide to a guanine nucleotide.

O superscript 6 end superscript-methylguanine has a five-membered ring on the right with N substituted for C at the top vertex at position 7, N substituted for C at the lower right vertex at position 9 and further bonded to R, a double bond between C 7 and C 8, and a double bond between C 4 and C 5 shared with a six-membered ring to the left. The six-membered ring has N substituted for C at its bottom right vertex at position 3, a double bond between position 3 and position 2, N substituted for C at position 1, C at the lower right vertex at position 2 bonded to N further bonded to 2 H, a double bond between positions 1 and 6, and C at the upper right vertex at position 6 double bonded to O above further bonded to red highlighted C H 3. An arrow labeled blue highlighted methyltransferase points right accompanied by a curved arrow from a blue oval bonded to C y s bonded to S H and labeled active to a blue oval bonded to C y 2 bonded to S bonded to red highlighted C H 3 and labeled inactive. This yields a guanine nucleotide. Guanine has a five-membered ring on the right with N substituted for C at the top vertex at position 7, N substituted for C at the lower right vertex at position 9 and further bonded to R, a double bond between C 7 and C 8, and a double bond between C 4 and C 5 shared with a six-membered ring to the left. The six-membered ring has N substituted for C at its bottom vertex at position 3, a double bond between position 3 and position 2, N substituted for C at position 1 and further bonded to H, C at the lower left vertex at position 2 bonded to N further bonded to 2 H, and C at the upper right vertex at position 6 double bonded to O above.

A two-part figure shows how D N A damage results in mutations by showing how O superscript 6-methylguanine mispairs with thymine in part a and how this can lead to G triple bonded to C to A double bonded to T mutations after replication. — FIGURE 25-26 Example of how DNA damage results in mutations. (a) The methylation product $O^{6}$ $upper O Superscript 6$ -methylguanine pairs with thymine rather than cytosine residues. (b) If not repaired, this leads to a $G \equiv C$ $upper G identical-to upper C$ to $A ═ T$ $upper A box drawings double horizontal upper T$ mutation after replication.

FIGURE 25-26 Example of how DNA damage results in mutations. (a) The methylation product $O^{6}$ $upper O Superscript 6$ -methylguanine pairs with thymine rather than cytosine residues. (b) If not repaired, this leads to a $G \equiv C$ $upper G identical-to upper C$ to $A ═ T$ $upper A box drawings double horizontal upper T$ mutation after replication.

Part a shows the structure of guanine with three hydrogen bonds to cytosine. Guanine has a five-membered ring on the left with N substituted for C at the top vertex at position 7, N substituted for C at the lower left vertex at position 9 and further bonded to R, a double bond between C 7 and C 8, and a double bond between C 4 and C 5 shared with a six-membered ring to the right. The six-membered ring has N substituted for C at its lower left vertex at position 3, a double bond between position 3 and position 2, N substituted for C at position 1 and further bonded to H, C at the lower right vertex at position 2 bonded to N further bonded to 2 H, and C at the upper right vertex at position 6 double bonded to O above. Cytosine is a six-membered ring with N substituted for C at the lower right vertex at position 1 and bonded to R, C 2 at the lower left vertex double bonded to O, N substituted for C at the left side vertex at position 3, C 4 at the upper left vertex bonded to N H 2, and double bonds between C 3 and C 4 and between C 5 and C 6. There are double bonds between O double bonded to C 6 of guanine and H bonded to N bonded to position 4 of cytosine, between H bonded to N at position 1 of guanine and N at position 3 of cytosine, and between H bonded to N bonded to position 2 of guanine and O double bonded to C 2 of cytosine. An arrow points down labeled methylation and replication. This yields O superscript 6 end superscript-methylguanine connected by two hydrogen bonds to thymine. O superscript 6 end superscript-methylguanine resembles guanine except that C 6 double bonded to O is bonded to O red highlighted C H 3 instead, N at position 1 is not bonded to H, and there is a double bond between C 1 and C 6. Thymine has N substituted for C at the lower right vertex at position 1 and bonded to R, C 2 at the lower left vertex double bonded to O, N substituted for C at the left side vertex at position 3, C 4 at the upper left vertex double bonded to O, C 5 at the upper right vertex bonded to C H 3, and a double bond between C 5 and C 6. There are hydrogen bond between H bonded to N at position 1 of O superscript 6-methylguanine and H bonded to N at position 3 and between H bonded to N bonded to position 2 of O superscript 6-methylguanine and O double bonded to C at position 2 of thymine. Part b shows a vertical double-stranded D N A molecule with a tan strand on the left and a blue strand on the right. Halfway along the molecule, G on the left is hydrogen bonded to C on the right. An arrow points right labeled methylation. This yields a similar molecule in which G is bonded to red highlighted C H 3 to the left. An arrow points down labeled replication. This yields a short blue vertical double-stranded piece of D N A that splits into left and right halves that have blue parent strands and tan daughter strands. The left half shows G in the blue strand bonded to red highlighted C H 3 to the left and hydrogen bonded to T in the tan strand to the right. The right half shows G in the tan strand hydrogen bonded to C in the blue strand. Arrows labeled replication point down from each one. The downward right-hand arrow from the right-hand daughter molecule points to text reading, correctly paired D N A (n o mutations). The left-hand arrow splits to show that two blue vertical molecules of D N A are produced. The left-hand molecule has G bonded to red-highlighted C H 3 to the left and hydrogen bonded to T to the right. The right-hand strand has A in place of G. This A s hydrogen bonded to T and enclosed in a bracket labeled mutation.

A very different but equally direct mechanism is used to repair 1-methyladenine and 3-methylcytosine. The amino groups of A and C residues are sometimes methylated when the DNA is single-stranded, and the methylation directly affects proper base pairing. In E. coli, oxidative demethylation of these alkylated nucleotides is mediated by the AlkB protein, a member of the $α {-ketoglutarate-Fe}^{2 +} - dependent$ $alpha hyphen ketoglutarate hyphen Fe Superscript 2 plus Baseline zero width space en-dash dependent$ dioxygenase superfamily (Fig. 25-27). (See Box 4-2 for a description of proline hydroxylation, catalyzed by another member of this enzyme family.)

A figure shows how alkylated bases can be directly repaired by A l k B by showing the conversion of 1-methyladenine to adenine and the conversion of 3-methylcytosine to cytosine. — FIGURE 25-27 Direct repair of alkylated bases by AlkB. The AlkB protein is an α-ketoglutarate- ${Fe}^{2 +}$ $Fe Superscript 2 plus$ –dependent hydroxylase (see Box 4-2). It catalyzes the oxidative demethylation of 1-methyladenine and 3-methylcytosine residues.

FIGURE 25-27 Direct repair of alkylated bases by AlkB. The AlkB protein is an α-ketoglutarate- ${Fe}^{2 +}$ $Fe Superscript 2 plus$ –dependent hydroxylase (see Box 4-2). It catalyzes the oxidative demethylation of 1-methyladenine and 3-methylcytosine residues.

1-methyladenine is a five-membered ring with N substituted for C at its lower left vertex at position 9 and further bonded, N substituted for C at position 7, a double bond between positions 7 and 8, and a double bond at its right side between positions 4 and 5 that is shared with the left side of a six-membered ring with N plus substituted for C at the upper right vertex at position 1 further bonded to red highlighted C H 3, N substituted for C at position 3 at the bottom vertex, a double bond between C 2 and C 3 at the lower right side, a double bond between C 1 and C 6 at the upper right side, and C 5 at the top vertex bonded to N H 2. An arrow points right above blue highlighted A l k B, nonhighlighted F e 2 plus and is accompanied by a curved arrow showing the conversion of O 2 plus alpha-ketoglutarate to C O 2 plus succinate. Alpha-ketoglutarate is a five-carbon vertical chain with C 1 at the bottom forming C O O minus; C 2 double bonded to C; C 3 and C 4 each boned to 2 H; and C 5 forming C O O minus. Succinate is a vertical four-carbon chain with C 1 at the bottom forming C O O minus; C 2 and C 3 bonded to 2 H; and C 4 forming C O O minus. This yields a structure that resembles 1-methyladenine except that N plus at the upper right vertex substituted for C at position 1 is bonded to red highlighted C H 2 bonded to nonhighlighted O H. An arrow points right accompanied by an arrow branching off to show the loss of formaldehyde, which is red highlighted H 2 C connected by a nonhighlighted double bond to O plus H plus. This yields adenine, which is similar to 1-methyladenine except that N at the upper right vertex at position 1 has no charge and is not further bonded. 3-methylcytosine is a six-membered ring with N substituted for C at the bottom vertex in position 1 and further bonded, N plus substituted for C at the upper right vertex at position 3 and further bonded to red highlighted C H 3, C 2 at the lower right vertex double bonded to O, C 4 at the top vertex bonded to C H 3, and double bonds between C 3 and C 4 and between C 5 and C 6. An arrow points right above blue highlighted A l k B, nonhighlighted F e 2 plus and is accompanied by a curved arrow showing the conversion of O 2 plus alpha-ketoglutarate to C O 2 plus succinate. This yields a similar molecule in which N plus substituted for C at position 3 is bonded to red highlighted C H 2 further bonded to O H. An arrow points right accompanied by an arrow branching off to show the loss of formaldehyde, which is red highlighted H 2 C connected by a nonhighlighted double bond to O plus H plus. This yields cytosine, which is similar to 3-methylcytosine except that N substituted for C at position 3 has no charge and is not further bonded.

The Interaction of Replication Forks with DNA Damage Can Lead to Error-Prone Translesion DNA Synthesis

The repair pathways considered to this point generally work only for lesions in double-stranded DNA, the undamaged strand providing the correct genetic information to restore the damaged strand to its original state. However, in certain types of lesions, such as double-strand breaks, double-strand cross-links, or lesions in a single-stranded DNA, the complementary strand is itself damaged or is absent. Double-strand breaks and lesions in single-stranded DNA most often arise when a replication fork encounters an unrepaired DNA lesion (Fig. 25-28). Such lesions and DNA cross-links can also result from ionizing radiation and oxidative reactions.

A figure shows the effects of types of D N A damage on D N A replication. — FIGURE 25-28 DNA damage and its effect on DNA replication. If the replication fork encounters an unrepaired lesion or strand break, the DNA polymerase sometimes disengages and re-initiates downstream. The lesion remains in an unreplicated, single-stranded gap that is left behind the replication fork (left). In other cases, a replication fork may encounter a lesion that is actively undergoing repair such that a transient break is present in one of the template strands. When the replication fork encounters it, the single-strand break becomes a double-strand break (right). In each case, the damage to one strand cannot be repaired by mechanisms described earlier in this chapter, because the complementary strand required to direct accurate repair is damaged or absent. There are at least two possible avenues for repair: recombinational DNA repair or, when lesions are unusually numerous, error-prone repair. The latter mechanism involves translesion DNA polymerases such as DNA polymerase V, encoded by the *umuC* and *umuD* genes and activated by the RecA protein that can replicate, albeit inaccurately, over many types of lesions. The repair mechanism is “error-prone” because mutations often result.

Two blue strands forming double stranded D N A extend up and down to the left to form a replication fork. An orange arrow runs beneath the top strand and points right into the replication fork. A short orange arrow runs along the bottom blue strand from the replication fork to the midpoint of the newly formed strand, where a second arrow points right to the left end of the strand. A black rectangle on the bottom blue strand just to the right of the replication fork is labeled, unrepaired lesion. An arrow points down to show a similar molecule in which the replication fork has moved to the right. The top orange arrow runs the length of the blue strand to point into the replication fork. The bottom strand still has a series of two orange arrows to the left, then has a break where only single stranded blue D N A is present with a black rectangle on the left side of this region. Another orange arrow begins int eh replication fork and points right to just before the black rectangle. An arrow points down to text reading, recombinational D N A repair or error-prone repair. On the right, a similar replication fork with double stranded blue D N A to the right is shown but there is a space in the top blue strand of the double-stranded D N A to the right of the replication fork. This is labeled unrepaired break. An arrow points down to show a short blue strand of D N A with an orange arrow pointing right beneath it along its entire length. Below, a longer blue strand is shown with a series of two arrows and a partial arrow pointing left. The end of the short blue strand above the orange arrow is labeled, double-strand break. An arrow points down to text reading, recombinational D N A repair.

At a stalled bacterial replication fork, there are two avenues for repair. In the absence of a second strand, the information required for accurate repair must come from a separate, homologous chromosome. The repair system thus involves homologous genetic recombination. This recombinational DNA repair is considered in detail in Section 25.3. Under some conditions, a second repair pathway, error-prone translesion DNA synthesis (often abbreviated TLS), becomes available. When this pathway is active, DNA repair is significantly less accurate, and a high mutation rate can result. In bacteria, error-prone translesion DNA synthesis is part of a cellular stress response to extensive DNA damage known, appropriately enough, as the SOS response. Some of the 40 or more SOS proteins, such as the UvrA and UvrB proteins involved in the error-free nucleotide-excision repair already described, are normally present in the cell but are induced to higher levels as part of the SOS response. Additional SOS proteins participate in the pathway for error-prone repair; these include the UmuC and UmuD proteins (unmutable; lack of the umu gene eliminates error-prone repair). The UmuD protein is cleaved in an SOS-regulated process to a shorter form called $UmuD'$ $UmuD prime$ , which forms a complex with UmuC and a protein called RecA (described in Section 25.3) to create a specialized DNA polymerase, DNA polymerase V $(UmuD'_{2} UmuCRecA)$ $left-parenthesis UmuD prime Subscript 2 Baseline zero width space UmuCRecA right-parenthesis$ , which can replicate past many of the DNA lesions that would normally block replication. Proper base pairing is often impossible at the site of such a lesion, so this translesion replication is error-prone.

Given the emphasis on the importance of genomic integrity throughout this chapter, the existence of a system that increases the rate of mutation may seem incongruous. However, we can think of this system as a desperation strategy. The umuC and umuD genes are fully induced only late in the SOS response, and they are not activated for translesion synthesis initiated by UmuD cleavage unless the levels of DNA damage are particularly high and all replication forks are blocked. The mutations resulting from DNA polymerase V–mediated replication kill some cells and create deleterious mutations in others, but this is the biological price a species pays to overcome an otherwise insurmountable barrier to replication, as it permits at least a few mutant daughter cells to survive. The resultant mutations contribute to evolution.

Yet another DNA polymerase, DNA polymerase IV, is also induced during the SOS response. Replication by DNA polymerase IV, a product of the dinB gene, is also highly error-prone. The bacterial DNA polymerases IV and V (Table 25-1) are part of a family of TLS polymerases found in all organisms. These enzymes lack a proofreading exonuclease and have a more open active site than other DNA polymerases, one that accommodates damaged template nucleotides. With these enzymes, the fidelity of base selection during replication may be reduced by a factor of $10^{2}$ $10 squared$ , lowering overall replication fidelity to one error in ∼1,000 nucleotides.

Mammals have many low-fidelity DNA polymerases of the TLS polymerase family. However, the presence of these enzymes does not necessarily translate into an unacceptable mutational burden, because most of the enzymes also have specialized functions in DNA repair. DNA polymerase $η$ $eta$ (eta), for example, found in all eukaryotes, promotes translesion synthesis primarily across cyclobutane T–T dimers. Few mutations result, because the enzyme preferentially inserts two A residues across from the linked T residues. Several other low-fidelity polymerases, including DNA polymerases β, $ι$ $iota$ (iota), and $λ$ $lamda$ , have specialized roles in eukaryotic base-excision repair. Each of these enzymes has a $5'$ $5 prime$ -deoxyribose phosphate lyase activity in addition to its polymerase activity. After base removal by a glycosylase and backbone cleavage by an AP endonuclease, these polymerases remove the abasic site (a $5'$ $5 prime$ -deoxyribose phosphate) and fill in the very short gap. The frequency of mutation due to DNA polymerase $η$ $eta$ activity is minimized by the short lengths (often one nucleotide) of DNA synthesized.

What emerges from research into cellular DNA repair systems is a picture of a DNA metabolism that maintains genomic integrity with multiple and often redundant systems. Most of the major DNA repair systems occur in all organisms. These repair systems are often integrated with the DNA replication systems and are complemented by recombination systems, which we turn to next.

SUMMARY 25.2 DNA Repair

Mutations are genomic changes that alter the information in DNA. When mutations occur in genes encoding enzymes involved in DNA repair, the loss of function can lead to cancer.
Cells have many systems for DNA repair. Major repair systems present in all organisms include mismatch repair, base excision repair, nucleotide-excision repair, and direct repair.
In bacteria, TLS DNA polymerases respond to very heavy DNA damage with error-prone translesion DNA synthesis. In eukaryotes, similar polymerases have specialized roles in DNA repair that minimize the introduction of mutations.