L9: Homology directed repair, homologous recombination

Question 1

Available templates in homology-directed DSB repair… when might recombination be applied?

Answer 1

• Sister chromatid (identical, late S-phase, G2)
• Homologous Chr. (G1, may result in LOH)

Homology-directed repair pathway is adapted for recombination - crossing over of Chr.s in meiosis, bacterial conjugation, transduction and transformation, and occasionally in mitosis

Question 2

Four phases processes conserved across organisms (in homology-directed repair)

Answer 2

1. Pre-synapsis: Generation of ssDNA at site of DSB
2. Synapsis: Pairing by one of ss ends invading an intact homologous duplex to form a heteroduplex
3. Post-synapsis: Repair of damaged duplex by DNA synthesis using strands from intact duplex as a template
4. Post-synapsis (ctd): Separation of 2 duplexes

Question 3

Pre-synapsis

Answer 3

ssDNA (~50 nts) generated at break site by helicases and exonucleases
-> end resection

Question 4

Synapsis

Answer 4

One of 3’ ends invades the intact duplex and bps, making a heteroduplex and forming a D-loop (pairing in D-loop is specific; ~90% accuracy over 20-30bp stretch

Question 5

SDSA process

Answer 5

Synthesis-dependent strand annealing
-The invading 3’OH end serves as a primer for DNA synthesis - D-loop migrates as new DNA synthesised
- Newly synthesised strand released from template strand when it can pair with the original 3’ overhang
- Following recapture of the end of the newly synth. strand, the damaged DNA now has a region of ssDNA then ds gap
- Gap filled w/ newly synth strand as template, nicks ligated

Question 6

SDSA characterised by:

Answer 6

• New DNA sans the break, both strands are the result of new DNA synthesis (conservative)
• No joining of DNA from undamaged duplex to repaired duplex

Question 7

Homology-directed repair (key proteins) - bacteria

Answer 7

• RecBCD: Helicase and nuclease activity
  -> Generates ssDNA tails at DSBs
• RecA recombinase: Binds to 3’ end, searches for homologous DNA, promotes strand invasion of 3’ tail to displace D-loop in a homologous duplex
• Rad51 replaces RecA in eukaryotes
  -> Rad51 is essential; KOs fatal

Question 8

RecBCD activity

Answer 8

• Attaches at DSB, moves along DNA, unwinding and degrading it until Chi sequence encountered (over-represented 8bp sequence, ~once per 5kbp)
• At this point, nuclease activity decreases and preferentially degrades 5’ strand
  -> 3’ ss tail left
• RecBCD facilitates RecA protein binding to ss tail
• recBCD mutants v. sensitive to DNA damaging agents

Question 9

RecA activity (homology-directed repair)

Answer 9

• Loaded onto ssDNA, forms pre-synaptic filament (helical nucleoprotein filament)
• This distorts the DNA, extending it by up to 50%
• Filament promotes pairing w/ homologous DNA

Question 10

RecA in vivo vs in vitro

Answer 10

• In vitro, RecA/Rad51 sufficient for strand exchange
• In vivo, other proteins involved SSB/RA prevent secondary structures impeding the reaction
• BRCA1 and BRCA2 are accessory proteins, defects in these predispose individuals to breast cancer

Question 11

Fidelity of homology-directed repair under normal circumstances

Answer 11

• Usually high fidelity, identical template (chromosome in bacteria, sister chromatid in eukaryotes (late S, G2))
• DNA w/ DSB restored to its pre-damage sequence

Question 12

Gene conversion (homology-directed repair in G1 phase)

Answer 12

• Homologous Chr. used as template in G1 when sister chromatid not available
• Usually slightly different in sequence
  -> repaired stretch now has sequence of the homolog (gene conversion)
  -> LOH

Question 13

Homologous recombination

Answer 13

• Uses same cellular machinery as homology-directed repair, but generates new combinations of genetic information
• Can occur between any DNA molecules with extensive regions of identical sequence or v. similar sequence
• Can result in reciprocal exchanges of large segments of DNA between homologous duplexes

Question 14

When does homologous recombination occur in eukaryotes, what are its key roles, and how is it initiated in this instance?

Answer 14

During meiosis I, plays key roles in…
- Providing repair mechanism for hom. Chr.s, physically connects recombining Chr.s ensuring separation of hom. Chr.s in Meiosis I
- Generating diversity by reciprocal exchange of random segments of maternal and paternal (non-sister) chromatids
-> unique gametes

Meiotic rec. initiated at DSBs, ‘Spo11’ introduces DSB to initiate it as Chr.s start to repair

Question 15

Homologous recombination process

Answer 15

1. End resection by RecBCD
2. Invasion of homologous duplex to form D-loop, by RecA
3. DNA synthesis from the 3’ end of the invading strand displaces more of the complementary strand, and the second end is captured by the D-loop
4. Semi-conservative DNA synthesis from both the invading strand and the captured strand
5. Two intact dsDNA regions joined by two Holliday Junctions are formed

Question 16

Holliday Junctions

Answer 16

• 4-armed, cross-stranded structures that can move along participating DNA molecules (physically linked by region of heteroduplex DNA and exchanged strands)
• Move by branch migration

Question 17

Branch Migration proteins and how they operate- Bacteria

Answer 17

• Driven by RuvAB complex
  -> tetramer of RuvA which binds to the 4 arms of H Junction, hexameric helicase of RuvB, w/ one hexamer binding to each of two opposite arms of junction
• RuvB moves H Junct. along DNA, breaking and re-forming bps in one direction
  ->planar, unfolded cross-like structure, resolved by placing symmetric nicks in either plane across junction
• RuvAB recruits RuvC ( H Junct. resolvase) to cleave junction, separating participating DNA molecules
  -> Direction of cleavage determines if no DNA is exchanged or if recombinant is produced (vertical or horizontal)

Question 18

Load strand invasion proteins

Answer 18

• Bacteria: RecFOR
• Euk: Rad52, BRCA2