Chromosome biology lecture 5 Flashcards
3 stages of recombination
- Initiation
- Synapsis (homologous strand paired + strand exchange occurs btw them)
- Post-synapsis (migration + resolution or dissolution)
Identification of recombination genes
1. Mutants w/ altered capacity for recomb
- 2 types of mutants predicted: x produced recomb in a cross or ↑ exchange btw genetic marker
- Cross used Her strain of E coli
- Hfr- (sensitive to streptomycin) w/ F- strain (Str resistant) ↑ mutation needed
- Recomb btw tDNA + F- cell → recomb F- resistant to Str that grow w/o Pro
- If mutant in F- cell ↓ efficiency of recomb, ↓ recombinants recovered
- Found ↓ recombination frequency than WT parent
- Repeat w/ different donor
Identification of recombination genes
2. Mutants defective in DNA repair
- Mutants that inactivate recomb genes make cell ↑ sensitive to killing effect of DNA damage
- RecA mutant = hypersistive to UV light
- RecB + C identified
- Double mutant, suppressor (mutations at new locus or mutations in 2 new loci)
Identification of recombination genes
3. Screen for relevant biochemical activities in fractionated cells
- Used an assay
- Run products on native + denaturing gels using radioactive labels + detect w/ autoradiography
- HJ resolves makes pair of symmetrical inversions close to junction
Identification of recombination genes
4. Identify similar structures to E already known
- Proteins that are well conserved = SbccD or Mre11-Rad50
Biochemistry of recombination
Initiation of HR
- ds break = resected, RecB,C,D
- RecB(3’-5’), RecD(5’-3’)
- Rapid degradation of 3’ end, less 5’
- At Chi, slows down (1000bp/s→150bp/s), conf change in RecB where switch to 5’ end degradation more
- Makes 3’ss tails w/ Chi site, RecA can bind + invade using 3’ end
Biochemistry of recombination
Homologous pairing + strand exchange
- RecA (e coli), Rad51 (eukaryotes)
- During HR, ssDNA protected by SSB in E coli/ RPA in humans
- RecA displaces + searches for homologous sequence
RecA structure
- Core domain w/ walker A+B
- ATP lies btw adjacent monomers, ATP bound form has ↑ affinity for DNA
- RecA-DNA needs to bind ATP to search for homologous DNA
Rad51 filament formation single molecule analysis
- DNA stained w/ YOYO1 dye
- Bead moved into buffer that stretches out DNA, then to another channel w/ fluorescently labelled Rad51, pulled back to buffer channel for visualisation
- After 18 mins, several Rad51 nucleation events
- 4/5 monomers of Rad51/RecA = nucleation unit
- Competition w/ SSB prevents unwanted recombination
- Dimer of RecA is minimal oligomer needed
RecA/Rad51 mediators
- RecA poorly competes w/ SSB
- Helped by RecF, RecO + RecR (E coli)
- RecFOR promotes recA nucleation on SSB coated ssDNA
- RecOR aids nucleation at other sites
- RecO traps DNA transiently released by ssDNA
- In yeast, mediators = Rad52 + Rad55-Ra57
- In humans, BRCA2 mediates filament formation, inhibits Rad51 ATPase
Strand exchange
- ATP binding needed for both
- Thought find homologous DNA by simple collision
- Filament has a weaker 2o DNA bs next to ssDNA, DNA randomly sampled for homology
- Minimum 8bp needed
Experiment
Strand exchange
- Circular ssdNA incubated w/ SSB, RecA + buffer
- RecA exchanges complementary DNA strand from the linear duplex to circular + ss linear molecule
3D homology search
- fluroescently labelled RecA interacts w/ bacteriophage ds DNA
- This DNA is dipped into reservoir containing ssDNA filaments then → observational chamber
- Can manipulate ds DNA
- Pairing of RecA to dsDNA = ↑ efficient when DNA is in tangled state
- Intersegmental contact sampling to search for homologous DNA
Biochemistry of recombination
Holliday junction branch migration + resolution
- D loop = resolved by cross over or non crossover by HJ resolvases like RuvC or Gen1
- Also can be processed w/ dissolution
- HJ move towards each other until collapse into hemicatenane
- Cleavage of 1 strand enables uncleared strand to pass through nick
- Nick resected by ligase
- RuvB anchored on DNA, translocates along DNA pumping out DNA across surface of RuvA (ATP hydrolysis)