DNA Replication and Repair

Know how abasic sites and deaminations are generated in DNA.

Abasic sites (AP sites): thousands of bases are spontaneously hydrolyzed from ribose in our body each day. Intact backbone, but no bases - no information. Deamination: cytosine undergoes a transamination reaction with water. Cytosine is then converted to uracil. Guanine ---> 8-oxoguanine: caused by reactive oxygen species such as peroxide and results in a G--->T transversion.

Know the characteristics and origins of cancer.

Cancer is characterized by uncontrolled cell division, and results from accumulated mutations. There are multiple diseases with multiple origins. Mutations can be induced by: 1. Environmental factors such as sun, smoke 2. Genetic predisposition, such as BRCA1 3. Infections, such as HPV, hepatitis B 4. Spontaneous mutations

Know why DNA incorporates thymine nucleotides instead of uracil.

DNA incorporates thymine instead of uracil so that deamination errors can be recognized before they are propagated. The U shouldn't be there and is easily recognized.

Know why DNA is negatively supercoiled.

DNA is always negatively supercoiled. This is necessary to compact the DNA into the cell and makes it easier to separate the strands. If DNA positively supercoiled, packing would be as efficient, but separation of strands would be much more difficult.

Be able to describe the structure and purpose of heterochromatin and euchromatin.

Euchromatin is lightly packed DNA (eg. beads-on-a-string) that is heavily transcribed while heterochromatin is tightly packed (eg. 30 nm fibre). Stains will heavily stain tighter packing.

Be able to describe the steps of DNA replication and the role of the following components: helicase, single-strand binding protein (SSB), type I and type II topoisomerases, primase, DNA polymerase (I, III, δ, and ε), telomerase, ligase, and RNase H.

Helicase uses ATP to unwind the DNA helix. SSB binds the single-stranded regions to prevent reannealing or nuclease digestion. Type I topoisomerase cuts a single strand, move the other strand through it to relieve tension. Type II topoisomerase cuts both strands. DNA polymerase adds deoxynucleotides to the free 3' OH end of an existing strand. E. coli has 5 polymerases (DNA pol I ---> DNA pol V). Pol III is the main polymerase for replication. Others may have repair functions. Humans have at least 12 (alpha, delta, epsilon, etc). Delta - lagging strand synthesis. Epsilon - leading strand synthesis. Primase introduces a short RNA primer that is complementary to the template strand. This gives us the initial 3' OH that we are going to extend. DNA ligase ligates two strands together - can do this either using ATP or NADH. DNA ligase. Seals break in sugar-phosphate backbone. Used to connect Okazaki fragments.

Know how DNA and histones may be covalently modified and how this affects gene expression.

Linear DNA is negatively supercoiled by wrapping the double helix around a core of 8 histones to form a nucleosome. Histones have a lot of lysines and arginines - they tend to be basic. They have to interact with DNA regardless of sequence, so they interact with the negatively-charged sugar-phosphate backbone. Having DNA wrapped around proteins makes it somewhat inaccessible for transcription machinery. How compact the DNA is affects how much it is expressed. Histones can be modified to adjust levels of expression, and the DNA itself can as well. Methylation of cytosine affects transcription greatly. Methylation of cytosine residues in CpG sequences inactivates genes. This is important for genetic imprinting, where either paternal/maternal gene is silenced. Covalent modification of histones may increase or repress transcription. For histone covalent modifications, these include acetylation, methylation, phosphorylation, and ubiquitination. Hard to guess how one specific modification is going to affect transcription. Acetylation is associated with increased transcription. DNA loosened from histones, so easier to transcribe. It is loosened because the acetyl group interacts with the lysine to depolarize it. DNA and histone modifications are important to epigenetics. Epigenetics: we're considering more than just the DNA sequence - changes in phenotype that do not involve altering the underlying DNA sequence.

Know what is meant by the proofreading activity of a polymerase and how it is accomplished along with the consequence for the fidelity of replication.

Most DNA polymerases have a proofreading function: the polymerase slows down due to the bulge caused by the mispairing and a 3' to 5' exonuclease removes the misincorporated nucleotide. Error rate of DNA pol without proofreading is 1 in 10,000 bases. Error rate of DNA pol with proofreading is 1 in 1,000,000 bases. Error rate with proofreading and DNA repair is 1 in 100,000,000. A 5' to 3' exonuclease such as RNase H or DNA pol I removes the RNA primer from each Okazaki fragment. DNA ligase forms a phosphodiester bond to join neighboring fragments.

Be able to show the arrow pushing mechanism of the DNA polymerase reaction.

Nucleophilic attack of the 3' OH on the alpha-phosphate of an incoming dNTP. 5' to 3' direction... Why? No chemical problem with going in reverse direction. However, if going in reverse direction, when we need to proofread (DNA polymerase), if a base needs to be excised and replaced, we would have to rephosphorylate the 5' end, which would be energetically unfavorable. The way it's set up, after the base is removed in proofreading, it can just continue to do the appropriate nucleophilic attack.

Be able to describe the basic structure and role of a nucleosome.

Nucleosome is DNA wrapped around 8 histones. This is a very conserved structure for eukaryotes.

Know what is meant by leading strand, lagging strand, Okazaki fragments why they are necessary.

Since DNA polymerase extends DNA in the 5' to 3' direction, one strand is synthesized continuously (the leading strand) and the other strand is synthesized in segments called Okazaki fragments (the lagging strand). A 5'to 3' exonuclease such as RNase H or DNA pol I removes the RNA primer from each Okazaki fragment. DNA ligase forms a phosphodiester bond to join neighboring fragments.

Know how the following repair mechanisms work and how they recognize errors in DNA: base excision repair, nucleotide excision repair, mismatch repair, and alkyltransferases.

Suicide proteins called alkyltransferases, transfer the methyl group (of a methylated base) to a cysteine (permanently inactivating the enzyme). This is energetically expensive. Base excision repair: cells have specific glycosylases that scan for and recognize common errors, such as T-G pairs, 8-oxoguanine, or uracil, and remove the incorrect base. Endonuclease cuts sugar-phosphate backbone and removes rest of that specific nucleotide. Then, DNA repair polymerase fills in correct base. Ligase seals sugar-phosphate backbone. Nucleotide excision repair: errors that significantly distort the helix are repaired by removing a segment of DNA surrounding the distortion. Example: pyrimidine dimers. If something's damaged, helicase separates the strands, then endonuclease removes nucleotides within region in question, then a DNA repair polymerase fills in the correct bases, and a ligase seals up the backbone. Mismatch repair: after replication, the mismatch repair system scans the newly synthesized strand and excises any segments with misincorporated nucleotides or knicks. Might degrade thousands of bases to get rid of the mismatch, so is energetically expensive, but worth it.

Describe how the ends of linear DNA are maintained.

Telomerase is a reverse transcriptase that carries an RNA template that codes for a new repeat while base-pairing with the previous repeat. To prevent important information from being lost, the enzyme telomerase adds repeating 5' TTAGGG 3' sequences to the 3' ends of chromosomes. At the 3' end of linear template DNA, DNA polymerase is unable to add new nucleotides as there is no free 3' OH. Consequently, linear DNA would shorten after each round of replication, so cells that must divide a lot use the enzyme telomerase to extend DNA. Telomerase has an associated RNA component that binds to the template DNA and also provides an RNA template to add DNA TTAGGG repeats to the end of the existing DNA template. These 6 nucleotide repeats are formed using the reverse transcriptase activity of the telomerase. Telomerase adds hundreds of these repeats to the template DNA, providing room for DNA polymerase to copy this template so that no essential DNA is lost during replication.

Be able to describe the structure and role of telomeres in cells.

The ends of chromosomes adopt special structures called telomeres where a repeating sequence of DNA folds into a loop and binds protective proteins. Linear DNA is susceptible to exonuclease digestion as well as random end-joining by the DNA repair machinery. Linear DNA also gets shorter with each round of replication since the 5' RNA primer cannot be replaced by DNA.

Know what is meant by transversion and transition point mutations.

Transversion: a mutation leading to a switch between a purine and a pyrimidine. Transition: purine to purine or pyrimidine to pyrimidine mutation.

Know how carcinogens such as UV light, benzopyrene, methylating agents, and reactive oxygen species induce DNA mutations.

UV light promotes pyrimidine dimers (most frequently between thymines). This brings them closer together, and disturbs the double helix structure. Some organisms have enzymes called photolyases which fix this problem. We have nucleotide excision repair. Smoke: Benzopyrene is oxidized in the liver and then forms an adduct with N2 of guanine, disrupts base pairing. Replication machinery misinterprets what guanine actually is - benzopyrene causes a G--->T transversion. Methylation causes transition (guanine to adenine, for example). Alters H-bonding capabilities such that base-pairing ends up being incorrect. What should be a G-C pair becomes a G-T pair. DNA is subject to oxidative damage (red arrows), spontaneous hydrolysis (blue arrows), and methylation (green arrows), so the cell must have mechanisms to recognize and repair damaged DNA. Electrons escape from ETC and create peroxides. These peroxides can modify biomolecules, like DNA.