BIOL 3500: DNA Repair Mechanisms

What are the steps of Homology-directed recombination repair?

*Starts with identical strand (division not complete yet) 1. One of the strands acquires a break - 2nd strand becomes the template 2. 5' to 3; exonuclease digests from the break, RAD51 protein as well as others bind ssDNA *RAD51 induces an intermediate to form "Holliday intermediate" 3. RAD51 bonded to ssDNA comes and invades the template and displaces one of the strands of the template strand (ssDNA invades sister chromatid at homologous sequence) 4. DNA polymerase extends invading strands in 5' to 3' direction (ssDNA acting as primer) 5. The released invading strand anneals with the remaining single-stranded region 6. DNA polymerase fills in gap 5' to 3' direction 7. DNA ligase seals any phosphodiester bond nicks

What are the steps of postreplicative gap repair?

1. Ahead of the replication fork there is a thymine dimer - DNA polymerase skips over it during replication so a gap is left where the dimer would be (lesion in template strand halts DNA synthesis and then synthesis is reinitiated downstream, leaving a ssDNA gap) 2. RecA is recruited in and coats the ssDNA (binds ssDNA) 3. RecA causes ssDNA to invade lower molecule (dsDNA) *ssDNA pairs with complement and displaces the newly synthesized strand 4. A portion of the template strand from lower molecule is transferred to the upper molecule - leaves ssDNA gap in lower molecule and fixes the top gap (lesion in upper molecule uses stolen strand as a template for repair) 5. Now the lower molecule gap is filled in using newly synthesized strand *easier to repair here than just repairing the original break bc this one doesn't have the dimer *Pics in notes - slides 27-29 *RecA doesn't do it all - recruits polymerase and ligase (so not direct repair)

What are the different basic types of excision repair? Are these direct or indirect?

1. Base excision repair (just takes out the one base) 2. Nucleotide excision repair (takes out the whole nucleotide) 3. Mismatch repair *Indirect

What are the basic types of DNA repair mechanisms

1. Damage reversal (direct repair) 2. Excision repair (main type that works with excise, alter, and repair) 3. Double-strand break repair 4. Postreplicative repair

Describe the complete process of prokaryotic methyl-directed mismatch repair

1. MutS finds mismatch (recognizes mismatch as a bulge in the DNA sequence) 2. MutH binds to the hemimethylated sequences 3. MutS/MutL complex binds MutH 4. Mut H cuts nonmethylated strand (upstream of the defect) 5. MutU unwinds strands (helicase activity) 6. 3' to 5' exonuclease digests nonmethylated strand (cuts where the cut was made and past MutS) 7. DNA polymerase fills vacant region (fills in unmethylated region) 8. DNA ligase seals ends

Describe the process of base excision repair. Which step is different for prokaryotes and eukaryotes and explain what happens in each.

1. N-glycosylase recognizes an abnormal base and cleaves the bond between the base and the sugar. 2. AP endonuclease recognizes a missing base and cleaves the DNA backbone on the 5′ side of the missing base. 3. This step is different for Euk. and Prok.; there is a nick in the backbone, now other enzymes must come in and fix it Prokaryotes: - E. coli DNA polymerase I uses its 5′ 3′ exonuclease activity to remove the damaged region, then fills in the region with normal DNA. DNA ligase seals the region. Eukaryotes: Have 2 choices 1) In eukaryotes such as humans, DNA polymerase β can remove the apyrimidinic nucleotide and replace it with the correct nucleotide. DNA ligase seals the region. 2) In eukaryotes such as humans, DNA polymerase δ or ε can synthesize a short segment of DNA, which generates a flap - Flap is removed by flap endonuclease. DNA ligase seals the region.

Describe the complete process of NER in E. coli. What is the same or different with this process in humans?

1. Protein trimer slides on DNA in search of damage (searches for the damage) - 2 UvrA - 1 UvrB 2. Damaged site identified: - 2 UvrA dissociate (leave - this was their only role) - UvrC comes in and binds to UvrB and produces an enzyme complex called the ABC excinuclease 3. UvrB & UvrC make incisions on both sides of lesion - UvrB likely does 3' cut **usually 4-5 nt's away from lesion - UvrC likely does 5' cut **usually 8 nt's away from lesion 4. UvrB & C released (cuts were single standed), UvrD binds - UvrD has helicase activity - unravels DNA at site - DNA pol. I fills in gap using undamaged strand as template 5. DNA ligase joins segments *In humans these are not the same enzymes (has complexes instead of enzymes) though the general process and end result is the same

What are the two types of radiation-induced mutations? What is ionizing radiation considered?

1. Single-stranded DNA breaks: usually not serious, easily repaired (commonly done by x-rays - NBD and ligase just seals) 2. Double-stranded DNA breaks: Difficult to repair, frequently cause lasting mutation (pieces of chromosome completely separate and causes issues when they need to divide) Ionizing radiation is a mutagen and a CLASTOGEN (causes chromosome breaks)

What happens to LexA once the DNA is repaired? What do sulA and sulB do and why are they included in the SOS repair mechanism?

1. There is no more ssDNA to activate the RecA* activity of RecA 2. RecA* is not there to activate LexA 3. Uncatalyzed LexA binds to the SOS box of the promoter 4. Transcription is represses and the SOS response is over *LexA is restored to dimer form and can now repress again sulA and sulB inhibit cell division - probably there to increase time to repair the DNA before the next cell division (The SOS response upregulates repair mechanisms and down regulates cell division)

What is vital to organism survival? How many of this does the bacteria contain? What do each of these do differently? How are these processes usually?

DNA repair systems are vital to organism survival Bacteria contain several different repair systems - even when 1 not functional, mutation rate increases - Ex. mutator strains Each repair system fixes specific types of DNA alterations DNA repair systems usually multi-step processes *Each of these mechanisms of repair has a subset of kinds of mutations it works on (they are all essential)

What is a 2nd mechanisms for radiation-induced mutations? How does it work? What cells does it act in?

Damage reversal - repair of alkylation O6-methylguanine methyltransferase - removes methyl or ethyl groups from alkylated G's - transferase - transfers alkyl group from base to Cys in transferase - suicide protein! *This alkyl transferase steals the methyl group from the dimer onto itself which inactivates the nucleotide - fixes the nucleotide but is a one time thing (this enzyme can't act again because it took on this methyl group) Works in E. coli - E. coli transferase gene expression is induced by DNA alkylation - E. coli cells can develop resistance to damage This also happens in human cells - we have this enzyme

What is the first repair mechanism for radation-induced mutations? How does it work? Do we have this same enzyme in humans?

Direct repair of T-T dimers is damage reversal - repair of dimerization Photolyase splits dimers - excitation with blue light - termed photoreactivation *This is done in prokaryotes - do it directly with enzymes which are excited by blue light and they cut the extra bonds We have the photolyase enzyme in humans, but it only cuts ssDNA not dsDNA

What are the 4 key enzymes required in the NER process in E. coli? What other machinery is needed? What is it involved in?

E. coli NER system requires 4 key proteins: 1.UvrA 2.UvrB 3.UvrC 4.UvrD Also DNA polymerase & DNA ligase Uvr - involved in ultraviolet light repair of T-T dimers ( it does more than just this, though) *Uvr = how they were 1st discovered and were named based on that

What is the 2nd mechanism of double-strand break repair? What does it do? What enzyme is involved in it? Why is this process considered time sensitive?

Homology-directed recombination repair Restores sequence using sister chromatid as template *Similar to Holliday intermediate in recombination RAD51 (related to RecA) Time sensitive because it needs to be in sister chromatid form to occur (the sister chromatids are used to help repair breaks)

How does E. coli develop a resistance to damage caused by UV light through alkylation? What also can get a resistance from it and what are the implications of it?

In E. coli can develop a resistance to damage from alkylation overtime because they increase the expression of the genes that encode for the O6-methylguanine methyltransferase enzyme. Cancer cells can also get a resistance from alkylation chemotherapy in this same way - the ones that increase this enzyme and have a resistance are the ones that reproduce so the cancer becomes immune to it - this type of chemotherapy needs to be coupled with an inhibitor

What sequence does the LexA protein bind to? What does it do? What interacts with it and what does it do?

LexA normally bound at promoter sites of genes with role in SOS Each gene has consensus sequence in promoter = SOS box - LexA binds SOS box of the promoter - 5'-CTGX10CAG-3' LexA represses expression of these genes until SOS response occurs When ssDNA activates RecA, RecA* interacts with LexA - interaction induces LexA autocatalytic activities (LexA dimer is split and can't repress) - gene expression no longer repressed and leads to the SOS response in E. coli

What is the mismatch repair mechanism? What are the proteins involved in E. coli? What is the mechanism dependent on?

Methyl-directed mismatch repair - similar to NER system, but different proteins E. coli proteins involved: 1. MutS (recognizes mismatch - bulge in the DNA sequence) 2. MutL (linker which binds MutS to MutH) 3. MutH (hemimethylation - this is what it recognizes) 4. MutU (helicase actvity to unwind strands) This repair mechanism is dependent on the presence of methylation on parent strand but not progeny strand - aka for Eukaryotes it is right after replication but before maintenance methylation (in Prokaryotes methylation is for something different)

What is NER? What cells does this function in? What types of damages does it repair? List out the basic steps

NER = nucleotide excision repair Found in both eukaryotes and prokaryotes Repairs different types of damage - T-T dimers - chemically modified bases - AP sites - etc Several nucleotides in damaged strand removed, intact strand used as template for resynthesis of normal strand 1. A base in the DNA is damaged so that it is not functional - this damaged DNA is recognized 2. The excision repair proteins excise the damaged base and some adjacent bases 3. DNA polymerase adds the correct bases by 5' to 3' replication of the short strand 4. DNA ligase seals *Like a surgeon removing a tumor - takes some cells around it to make sure its all out

What is the 1st mechanism of double-strand break repair? What enzymes recognize this? Describe the basic process.

Non-homologous end joining (pieces get randomly put together) *Repair that is not a perfect fix - puts it back together but is left with mutations Ku = Ku70 + Ku80 (recognizes the damage, smooths the edges, and recruits kinase) *Nonspecific trimming and ligation 1. Uncut DNA strand 2. DNA strand break (with jagged edges) 3. Enzyme Ku heterodimer recognizes the break - Ku70 and Ku80 trim the ends of the break so they are smooth 4. Recruits in kinase 5. Kinase makes sure 5' end is phosphorylated so ligase can put it back together 6. DNA ligase seals

What is postreplicative gap repair? What enzyme catalyzes it? What is another name for it? How does the size of ssDNA and dsDNA compare?

Protects newly forming strand from mutations - T-T dimers can prevent proper base pairing during replication - can result in deletions Repair catalyzed by RecA protein Postreplicative gap repair = recombination repair ssDNA is a huge concentration of proteins including RecA so it is not necessarily smaller than dsDNA because ssDNA is attracted by so many proteins (For example, RecA coats ssDNA and this is larger than dsDNA so it is noticed by repair mechanisms)

What is an E. coli specific example of the postreplicative gap repair? What does it not involve? How does the filling of bases occur? Why do they do this?

SOS response in E. coli (Replication fork passes by, gaps left behind may be lethal; SOS response = EMERGENCY!) Does not involve recombination (randomly puts in bases) Bases are filled in without regard to proper pairing - many mismatches - may allow cell to survive & repair mismatches - may even create sequence making cell more viable in stressful environment (these mismatches (rarely) can lead to a beneficial mutation and give it a function it didn't have before) This is done in emergency situations because gaps can be lethal so the first priority is to fill in the gaps and then they can potentially go repair the mutations (not ideal - many mismatches, but a quick way to fix a big problem and then will use other repair mechanisms later)

How is the E. coli SOS response turned on? What does RecA do in the presence of ssDNA? Where is this protein normally? What genes are involved in the SOS response and why is this protein bound here?

SOS response turned on by: - excessive UV light exposure - mutagens In presence of ssDNA (aka a break or gap), RecA interacts with LexA protein - LexA product of lexA locus LexA normally bound at promoter sites of genes with role in SOS - 18 genes, including self - normally low level of basal expression - 18 genes include: recA, uvrA, uvrB, uvrD (NER genes) sulA & sulB - inhibit cell division *LexA is normally bound at the promoter of these genes and inhibits the expression of proteins involved in the SOS response until it is needed

How does UV light effect DNA? What do these do? What is this type of mutation called? What is it an example of and what cells is it in?

UV is relatively weak - cross-links adjacent pyrimidines on same DNA strand - forms thymine dimers *UV light makes photoproducts including thymine dimers which forms a cyclomutate and kink in the DNA strand (confuses polymerase during replication) Blocks DNA replication - machinery cannot recognize which bases to insert opposite the dimer - replication can continue if base added at random Radiation-induced mutations (an example of direct repair - in general Euk. doesn't have it so mostly in Prok.)

With UV-irradiated DNA, what are the altered DNA constituents called? What are the 2 prominent dimers seen? What type of UV radiation is most damaging to DNA? What does this explain the link between

UV-irradiated DNA - Many altered DNA constituents = photoproducts Prominent dimers: 1.Cyclobutane T dimer 2. 6,4 photoproduct T dimer (not a full ring but dimers still attached) *In either case these alterations cause an issue with replication UV radiation at 260 nm is most damaging to DNA - wavelength absorbed most strongly by DNA - abundant in sunlight Explains link between sun exposure & skin cancer

What Eukaryotic proteins involved in mismatch repair are homologous with Prokaryotic enzymes? What happens when human mismatch repair mechanisms are nonfunctional? What percentage of these mutations occur with each enzyme?

hMSH2 is homologous to MutS hMLH1, hPMS1, hPMS2 are homologous to MutL No mismatch repair mechanism leads to Lynch syndrome - mutator genes - hereditary nonpolyposis colon cancer (results from the defect of this mismatch repair causing them to build up more frequently than they would) - MLH1 = 50% (90-95% of them are due to sequence variants and 5-10% are due to deletions or rearrangements - MSH2 = 40% (50-80% caused by sequence variants and 17-50% are due to deletions or rearrangements) - MSH6 = 7-10% - PMS2 = <5%