Ch. 13 Recombinational DNA Repair and Homologous Recombination Flashcards
What can recombination between homologous DNA sequences do? (2)
- It can repair single and double strand chromosome breaks
- It can generate diversity in homologous chromosomes
In what cell classes does homologous recombination occur? (2)
It occurs in prokaryotes and eukaryotes.
How did homologous recombination likely come about?
Likely evolved as a DNA repair mechanism PRIOR to divergence of prokaryotes and eukaryotes.
How did homologous recombination move beyond mere DNA repair?
Evolution of diploidy allowed for a subsequent role in generating genetic diversity during gamete formation.
Why do double stranded breaks need to be repaired?
They are often lethal if un-repaired.
How can breaks occur? (3)
- replication forks can encounter a ss break, which can occur from a DNA lesion
- exposure to radiation
- exposure to other factors that break phosphodiester bonds
What can happen if there is a lesion and repair has NOT begun yet? (3)
- translesion synthesis may occur, and recombination isn’t needed
- the fork may stall until the repair completes
- the replisome may be blocked but restart downstream of the lesion
If the replisome is blocked by by a lesion that has not been repaired and it jumps downstream, which strand is it more likely to occur in?
More likely to happen in the lagging strand.
What can happen if there is a lesion and repair has begun, but not completed? (1)
A double stranded break will occur at the single strand break for one of the new helices.
During homologous recombination repair of ds breaks, what happens first?
The ends of DNA are degraded by nucleases and the strands are separated by helicases (RecBCD Complex). 5’ ends are degraded further and 3’ extensions/overhangs are created.
During homologous recombination repair of ds breaks, what happens second?
Another enzyme will bring the homologous DNA close to the broken chromosome.
During homologous recombination repair of ds breaks, what happens third?
RecA mediates strand invasion and formation of the D loop.
How does invasion and formation of the D loop occur? (3)
- a portion of a strand from an intact homologous chromosome is displaced from the intact chromosome (D loop formed)
- a 3’ overhang from the broken strand replaces the displaced DNA from the intact chromosome through base pairing
- multiple RecA subunits bind to mediate further exchange along the chromosomes; called branch migration
During homologous recombination repair of ds breaks, what happens fourth?
The 3’ ends of strands are extended using bases in the intact chromosome.
During homologous recombination repair of ds breaks, how can separation of the two chromosomes occur? (2)
- synthesis-dependent strand annealing
- double strand break repair
What is synthesis-dependent strand annealing and how does it occur (2)?
A way to separate chromosomes during HR.
1. Helicases break H-bonds between the invading strands, and the strands re-anneal.
2. Pol continues filling in gaps if needed and ligase seals nicks.
What is double strand break repair and how does it occur?
A way to separate chromosomes during HR break repair.
1. extension and ligation occurs while the helices are interconnected to create a Holliday junction
2. Holliday junctions are separated by specialized enzymes called resolvases
How can a Holliday junction be resolved? (2)
With crossover, or with no crossover.
What is the mechanism of resolving Holliday junctions? (3)
- Ruv proteins mediate resolition depending on which position they cut in the Holliday junction (X or Y)
2a. Cutting both outside strands, or both inside strands, leads to a non-crossover event with the original MOSTLY intact
2b. Cutting one outside strand and one inside strand leads to a crossover event (recombination)
What does the location the Ruv proteins cut at depend on?
The protein orientation on the DNA.
Where does homologous recombination happen in eukaryotes?
It occurs in meiosis I and allows for exchange between homologous chromosomes.
How does homologous recombination begin in meiosis I?
Double strand breaks are introduced early in prophase I at somewhat unpredictable regions of a chromatid.
What enzyme carries out HR in yeast?
Spo11. It is regulated!
How does Spo11 carry out HR? (3)
- a tyrosine residue acts as a nucleophile (e- donor) to break phosphodiester bonds
- endonucleases chew back the ends of the chromosomes to create 3’ extenstions
- RecA-equivalent proteins initiate double stranded break repair through homologous recombination
