Chromosome biology lecture 5 Flashcards

1
Q

3 stages of recombination

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

Identification of recombination genes

1. Mutants w/ altered capacity for recomb

A
  • 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
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3
Q

Identification of recombination genes

2. Mutants defective in DNA repair

A
  • 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)
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4
Q

Identification of recombination genes

3. Screen for relevant biochemical activities in fractionated cells

A
  • 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
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5
Q

Identification of recombination genes

4. Identify similar structures to E already known

A
  • Proteins that are well conserved = SbccD or Mre11-Rad50
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6
Q

Biochemistry of recombination

Initiation of HR

A
  • 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
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7
Q

Biochemistry of recombination

Homologous pairing + strand exchange

A
  • RecA (e coli), Rad51 (eukaryotes)
  • During HR, ssDNA protected by SSB in E coli/ RPA in humans
  • RecA displaces + searches for homologous sequence
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8
Q

RecA structure

A
  • 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
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9
Q

Rad51 filament formation single molecule analysis

A
  • 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
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10
Q

RecA/Rad51 mediators

A
  • 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
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11
Q

Strand exchange

A
  • 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
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12
Q

Experiment

Strand exchange

A
  • Circular ssdNA incubated w/ SSB, RecA + buffer

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

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13
Q

3D homology search

A
  • 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
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14
Q

Biochemistry of recombination

Holliday junction branch migration + resolution

A
  • 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)
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