Title: Jack Sparrow Biochem / Biochemistry / Biochemistry Chapter 6
Tags: #JackSparrow::#B/B::#Biochemistry::#Chapter.6

Q: What are the two forms of nucleic acids in the eukaryotes?
A: 1.  Deoxyribonucleic acid (DNA)
2.  Ribonucleic acid (RNA)

Q: What is a nucleoside?
A: A nucleoside is a 5 carbon sugar (pentose) bonded at Carbon 1 to a nitrogenous base by a covalent bond.
MNEMONIC: Side - Sugar and BaSe

Q: What is a nucleotide?
A: A nucleotide is a nucleoside with one or more phosphates attached to Carbon 5 of the sugar.  Often these molecules are named for the nitrogenous base and the number of phosphates (ATP is adenosine triphosphate!).  Nucleotides are the building blocks of DNA.
Refer to the image below for a good mnemonic.

Q: Why are nucleotides high energy molecules?
A: Almost all of the energy stored in nucleotides comes from the phosphate groups.  The presence of negatively charged phosphates next to one another results in large repulsion and high potential energy locked in the molecule.

Q: What is the difference between ribose and deoxyribose?
A: Ribose is a 5 carbon saccharide.  Deoxyribose is very similar, it just has an -H instead of an -OH on carbon 2.

Q: What are the names of the 5 nitrogenous bases of which DNA and RNA are collectively built?
A: 1.  Adenine
2.  Guanine
3.  Cytosine
4.  Thymine - DNA only
5.  Uracil - RNA only

Q: Describe the backbone of DNA.
A: The backbone of DNA is composed of alternating sugar and phosphate groups.  These sugars and phosphates are connected with 3'-5' phosphodiester bonds.  That is to say, a phosphate group links the 3' carbon of one sugar to the 5' carbon of the next sugar through a phosphate bond.
Remember, phosphate has a negative charge, so DNA and RNA strands are actually pretty negatively charged overall.

Q: Describe the polarity of DNA.
A: DNA as a long linear molecule that is considered by the enzymes that work on it to "start" with the 5' end and "end" with the 3' end.  Because of the one way nature of DNA enzyme activity, DNA is said to have polarity.

Q: How do we write the base sequence of a DNA or RNA strand?
A: DNA base sequences are written from 5' to 3' (left to right) using one letter abbreviations for the nitrogenous bases present.  For example, 5'-ATG-3' would be how you write the base sequence for a DNA molecule consisting of those three nucleotides.  If you are given a DNA base sequence that is written 3' to 5', it might not be a bad idea to flip it so it's easier to compare to other base sequences.

Q: Generally speaking, DNA is [single or double stranded?]
A: double stranded

Q: Generally speaking, RNA is [single or double stranded?]
A: single stranded

Q: What does it mean when we say the two strands of DNA are antiparallel?
A: Remember, the 5' to 3' directionality is very important for the enzymes interacting with DNA.  Antiparallel refers to the fact that the 5' to 3' direction for one strand is opposite of the other one.

Q: What are the two classes of the nitrogenous bases that make up DNA?
A: 1.  Purines: Adenine and Guanine
2.  Pyrimidines: Cytosine, Uracil, and Thymine
MNEMONIC:  CUT the PYe. The other two (A and G) are the purines.

Q: What are the nitrogenous bases?
A: The nitrogenous bases are a collection of nitrogen containing aromatic bases.  These bases make up the interior fundamental language of DNA.

Q: What are the main components of a DNA strand?
A: 1.  Phosphate groups in the backbone
2.  Deoxyriboses in the backbone
3.  Nitrogenous bases in the middle.

Q: What is the Watson-Crick model of DNA structure?
A: 1.  The two strands of DNA are antiparallel.  When one strand has polarity 5' to 3' down the page, the other has 5' to 3' up the page.
2.  The sugar Phosphate backbone is on the outside of the helix and the nitrogenous bases are on the inside
3.  The bases only pair with complementary bases via hydrogen bonds.  A always goes with T and C always goes with G
4.  Because of the specific pairing, the amount of A's is always equal to the amount of T's nad the amount of C's is always equal to the amount of G's

Q: What are major and minor grooves?
A: Major and minor grooves are grooves in DNA that arise as a result of the double helix.  These grooves are used as binding sites by proteins.

Q: What does it mean to denature DNA?
A: Denaturing DNA is when you disrupt the hydrogen bonds holding the bases together and the two strands of DNA separate.

Q: What does it mean to reanneal DNA?
A: Reannealing DNA is when DNA is brought back together after it has been denatured.

Q: What is probe DNA?
A: Probe DNA is a strand of DNA with a known sequence.  It is used in various testing and laboratory applications.

Q: What is a nucleosome?
A: A nucleosome is the name for a histone with DNA wrapped around it.

Q: What are the two types of chromatin?
A: 1.  Heterochromatin
2.  Euchromatin

Q: What is Heterochromatin?
A: Heterochromatin refers to DNA that is wrapped tightly around histones.  Heterochromatin is dark when viewed under microscope and is transcriptionally silent, meaning RNA polymerase cannot interact with Heterchromatin to make mRNA.

Q: What is Euchromatin?
A: Euchromatin refers to DNA that is wrapped loosely around histones.  Euchromatin is light when viewed under microscope and is transcriptionally active, meaning RNA polymerase can interact with euchromatin to make mRNA.

Q: Which base pair is stronger, A-T or C-G?
A: C-G is stronger because it is composed of three hydrogen bonds instead of just two.

Q: What is the replisome?
A: The replisome (aka replication complex) is a set of special proteins that assist DNA polyemerases to copy DNA.

Q: What is the origin of replication?
A: The origin of replication is where the DNA starts to unwind during copying.

Q: Generation of new DNA proceeds in [one direction or both directions?]
A: both directions

Q: How does replication of DNA proceed differently in prokaryotes versus eukaryotes?
A: Eukaryotes will have multiple origins of replication and result in two attached sister chromatids.
Prokaryotes will only have one origin of replication and result in two unattached chromosomes.

Q: What is the role of Helicase?
A: Helicase is an enzyme responsible for unwinding DNA, generating two single stranded templates ahead of the polymerase.

Q: What is the role of single stranded DNA binding proteins?
A: SSB's bind to unraveled DNA to prevent the reassoaciation of the DNA and to protect the base pairs from enzymes.

Q: What is supercoiling?
A: Supercoiling is the process of DNA wrapping on itself and getting tangled during replication.

Q: What is the role of topoisomerase?
A: Topoisomerase is an enzyme that alleviates the tension in the DNA in front of helicase so it doesn't supercoil or break.  It does so by temporarily cutting the strands to relieve tension and then putting them back together when they aren't in danger anymore.

Q: What is the parent strand?
A: The parent strands are the strands of original DNA that serve as the template for the replication of DNA.

Q: What is a daughter strand?
A: A daughter strand refers to the newly synthesized DNA strand.

Q: What does it mean when we say DNA replication is semiconservative?
A: Semiconservative means that you conserve some of the DNA when you copy it.  This is evidenced by the fact that each new DNA strand will have one parent strand (conserved) and one new daughter strand.

Q: What is DNA polymerase?
A: DNA polymerase is the class of enzymes that is responsible for reading the parental strand and synthesizing the new daughter strand.

Q: Describe the 5' to 3' directionality of DNA polymerase?
A: DNA polymerase reads DNA in the 3' to 5' direction (weird!).  However, it synthesizes DNA in the 5' to 3' direction.  This is the way it has to be so that daughter strand is antiparallel to the parent strand.

Q: What is the leading strand?
A: The leading strand is the daughter strand that is copied in a continous fashion because it is moving in the same direction as the replication fork.

Q: What is the lagging strand?
A: The lagging strand is the daughter strand that is copied in the direction opposite of the replication fork.  The fact that the DNA is being synthesized in the opposite direction of the replication fork is a huge deal and the body has had to evolve the okazaki fragment approach to synthesizing the lagging strand correctly.

Q: What are okazaki fragments?
A: Okazaki fragments refer to the short fragments of DNA that are discontinually produced and joined together to make the lagging strand.

Q: What are the 7 basic steps to DNA replication?
A: 1.  Helicase unwinds the DNA
2.  Single Stranded Binding Proteins protect the free DNA
3.  Primase synthesizes RNA primers to attract DNA polymerase
4.  DNA Polymerase binds and synthesizes DNA
5.  RNA primers are removed
6.  DNA Ligase joins Okazaki Fragments
7.  Topoisomerase reduces tension in the advancing coil

Q: The leading strand requires [one or many primers?]
A: one
Extra: You just put the first primer down and then continously synthesize the DNA until its done.
NOTE: primers shown in green.

Q: The lagging strand requires [one or many primers?]
A: many primers
Extra: Each individual okazaki fragment will actually require its own RNA primer so DNA polymerase can bind.

Q: What is Primase?
A: Primase is an enzyme that lays down RNA primers for DNA polymerase to bind to.

Q: How are RNA primers removed in Prokaryotes versus Eukaryotes?
A: RNA primers are removed in prokaryotes by DNA polymerase I.
RNA primers are removed in Eukaryotes by RNase H.

Q: What is DNA ligase?
A: DNA ligase is a molecule that can join together fragments of unconnected DNA.  DNA ligase joins okazaki fragments together!

Q: How is the DNA polymerase in prokaryotes and eukaryotes different?
A: In prokaryotes, DNA is replicated by DNA polymerase III.
In Eukaryotes, DNA is replicated by DNA polymerase alpha, delta, and epsilon.

Q: What are the 5 classic DNA polymerases in Eukaryotic cells?
A: Alpha, Beta, Gamma, Delta, and Epsilon.

What is the combined role of DNA pol Alpha, Delta, and Epsilon?

What is the role of DNA polymerase Gamma?

What is the combined role of DNA pol Beta and Epsilon?

What is the combined role of DNA pol delta and epsilon?

Q: What are the classic prokaryotic DNA enzymes?
A: 1.  DNA pol I
2.  DNA Pol II
2.  DNA Pol III

In what ways are the enzymes for DNA Replication similar and different in Prokaryotes versus eukaryotes?

Q: What are oncogenes?
A: Oncogenes are pieces of DNA that, if damaged (mutated) cause cancer.

Q: What type of genetic info is more likely to be an oncogene?
A: Most of the oncogenes are genes that have to do with cell-cycle and division timing.  Your eye color gene, on the other hand, probably wont give you cancer if it mutates.

Q: What are antioncogenes?
A: Antioncogenes are genes that stop and slow tumor progression.  This can be because they prevent mutations to DNA in the first place, fix them after they happen, or kill cancer cells.  When antioncogenes are mutated, they lose their anti-tumor properties.

Q: What are the two main ways DNA polymerase prevents errors in replication?
A: 1.  Proofreading
2.  Mismatch Repair

Q: Describe how DNA polymerase proof-reads bases.
A: Part of the DNA polymerase enzme is specifically designed to proofread.  When complementary strands have incorrectly paired bases the hydrogen bonds are unstable.  The proofreading section of DNA polymerase detects the unstable hydrogen bond and removes the incorrect base.

Q: Describe Mismatch Repair
A: In the G2 phase of the cell cycle, special enzymes will check the DNA for errors and correct them.

Q: What are the two main repair mechanisms of mismatch repair?
A: 1.  Nucleotide Excision Repair
2.  Base Excision Repair

Q: Describe Nucleotide Excision Repair?
A: In nucleotide excision repair, a section of a damaged DNA strand is completely cut out, phosphodiester back bone included, and then DNA polymerase fills in the gap, using the undamaged strands as a template.  The fixed strand is sealed back into place with DNA ligase.

Q: Describe Base Excision Repair?
A: In base excision repair, a damaged base is detected and removed by a glyosylaze enzyme, leaving behind an empty backbone called an AP site.  AP endonuclease recognizes the empty backbone and cuts it out.  DNA polymerase can then synthesize a complementary strand using the undamaged strand.  DNA ligase seals the repaired strand back into place.

Q: What are restriction enzymes?
A: Restriction enzymes (also known as restriction endonucleases) are enzymes that recognize palindromic double stranded DNA sequences and cut them.  These enzymes are isolated from bacteria and are used to cut up DNA strands in the laboratory.
A palindromic sequence of double stranded DNA is a strand of DNA that has the same 5' to 3' sequence on both strands, they're just going in opposite directions.

Q: What is a vector in the context of DNA Biotechnology?
A: A vector is usually a bacterial or viral plasmid that can be transferred to a host bacterium.

Q: What is a recombinant vector in the context of DNA Biotechnology?
A: A recombinant vector is a vector that has been modified with new DNA.

Q: How do you make a recombinant vector?
A: First, you use a specific restriction endonuclease to cut a certain piece of the vector's DNA out.
Then, you use the same specific restriction endonuclease to cut a piece of the DNA you are interested in out.
Because you used the same endonuclease, the foreign DNA will be able to fit the hole made in the vector DNA.
Finally, you just mix the foreign DNA fragment with the vector, mix them in annealing conditions and treat with DNA ligase to lock them into place.

Q: What is the point of making a recombinant plasmid vector?
A: When you put a little piece of DNA you are interested in into a recombinant vector, you can insert it into a bacterium.
The bacterium will grow, and in so doing will replicate its DNA, including the recombinant vector.
By letting big colonies of bacteria grow with your DNA you can generate large quantities of it.
Once you feel like you have enough bacteria, you can either kill the bacteria and re-treat them with the endonucleases to collect all the DNA the bacteria made for you or you can make them express the gene so you can collect all the protein that gets made from that DNA. (this is how we make insulin, we grow it in bacteria!)

Q: What are DNA libraries?
A: DNA libraries are large collections of known DNA sequences that are cloned into vectors for use in future studies.  DNA libraries can be Genomic or Complementary

Q: What is the difference between a Genomic Library and a Complementary (expression) Library?
A: A genomic DNA library contains large fragments of DNA that are digested out of DNA itself, which means these strands of DNA include both coding (exon) and noncoding (intron) regions.  This method is preferred for sequencing the entirety of an organism's genome.
A complementary DNA library is constructed by reverse-transcribing processed mRNA, which means these strands only contain the exons of the DNA from which they came.  This method is generally preferred for sequencing specific genes to identify their function.  It is also the only way to make transgenic animals and express genes in animals from which the DNA did not originate.

Q: What is Hybrdiziation?
A: Hybridization is the process of bringing two complementary base pair sequences together.  This can be DNA-DNA recognition or DNA-RNA recognition.

Q: Describe PCR
A: PCR is an a process that can produce millions of copies of a DNA sequence without using bacteria.
During PCR, DNA of interest is placed in a solution of nucleotides, denatured (heated up), replicated with custom made primers (you usually have to buy these for a project), and then cooled back down.  Each round of PCR doubles the amount of DNA, so it only takes a few rounds before you have tons of DNA.

Q: Describe Gel electrophoresis for DNA
A: DNA is negatively charged, and therefore will migrate towards the positive anode of the electrolytic cell.  Larger DNA will migrate slower, just like in protein electrophoresis.  Agarose is the preferred gel for DNA gel electrophoresis.

Q: What is a southern blot?
A: A southern blot is used to detect the presence and quantity of various DNA strands in a sample.  In this method, DNA is cut by restriction enzymes in specific ways and then separated by gel electrophoresis.  The separated DNA fragments are then transferred to a membrane where they are exposed to a probe.  The probe is radioactive single stranded DNA sequence that is visible under x-ray.
If the sequence in the DNA and the probe match up, they will hybridize, and that band of DNA on the membrane will be visible.

Q: How does DNA sequencing work?
A: DNA sequencing uses dideoxyribonucleotides (ddATP, ddGTP, etc.) to determine the sequence of a segment of DNA.
Dideoxyribonucleotides don't have a hydroxyl at Carbon 3, and will end DNA replication when they are incorporated into the DNA.  Eventually the sample will be composed of a bunch of fragments that end in the ddnucleotide of interest.  The ddnucleotide is easily read, and so you can separate the fragments based on what ddnucleotide they end in.  Then, using gel electrophoresis, you can see how long the chain was when the ddnucleotide was incorporated to determine the base order.

Q: What is gene therapy?
A: Gene therapy is a therapy for genetic diseases that involves replacing mutated or inaccurate copies of genes in humans with normal copies.
Modified Viruses are usually used to deliver the working gene to the host's DNA.

Q: What are transgenic mice?
A: Transgenic mice are mice that had a cloned gene insterted into their zygote or some other embryonic stem cell, meaning they have the gene in all of their adult cells.  Transgenes usually carry genetic diseases so that we can study the affects of the disease from early embryonic develoment through adulthood.

Q: What is a knockout mouse?
A: A knockout mouse is a mouse that had one of its genes removed as a zygote, meaning it lacks the gene completely as an adult.  Knockout mice are used to study the importance and effects of different genes.

Q: The more GC pairs in a DNA strand the [higher or lower] the denaturation temperature
A: higher
Extra: More hydrogen bonds means more intermolecular forces, which means higher denaturation temperature.