Chromosome biology lecture 5

Question 1

3 stages of recombination

Answer 1

1. Initiation
2. Synapsis (homologous strand paired + strand exchange occurs btw them)
3. Post-synapsis (migration + resolution or dissolution)

Question 2

Identification of recombination genes

1. Mutants w/ altered capacity for recomb

Answer 2

• 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

Question 3

Identification of recombination genes

2. Mutants defective in DNA repair

Answer 3

• 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)

Question 4

Identification of recombination genes

3. Screen for relevant biochemical activities in fractionated cells

Answer 4

• 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

Question 5

Identification of recombination genes

4. Identify similar structures to E already known

Answer 5

• Proteins that are well conserved = SbccD or Mre11-Rad50

Question 6

Biochemistry of recombination

Initiation of HR

Answer 6

• 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

Question 7

Biochemistry of recombination

Homologous pairing + strand exchange

Answer 7

• RecA (e coli), Rad51 (eukaryotes)
• During HR, ssDNA protected by SSB in E coli/ RPA in humans
• RecA displaces + searches for homologous sequence

Question 8

RecA structure

Answer 8

• 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

Question 9

Rad51 filament formation single molecule analysis

Answer 9

• 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

Question 10

RecA/Rad51 mediators

Answer 10

• 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

Question 11

Strand exchange

Answer 11

• 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

Question 12

Experiment

Strand exchange

Answer 12

• Circular ssdNA incubated w/ SSB, RecA + buffer

- RecA exchanges complementary DNA strand from the linear duplex to circular + ss linear molecule

Question 13

3D homology search

Answer 13

• 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

Question 14

Biochemistry of recombination

Holliday junction branch migration + resolution

Answer 14

• 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)