13. Biochemistry of homologous recombination Flashcards
What are the two pathways to repair DNA by homologous recombination in E. coli?
2 pathways to load RecA onto DNA and repair by homologous recombination:
- RecFOR for ss gaps
- RecBCD for ds breaks
What is the goal of RecFOR and RecBCD?
To load RecA - nucleoprotein filament that searches for homologous chromosome sites
What are the events after which a cell carries homologous recombination?
Homologous recombination carried after:
- dsDNA break repair
- dsDNA fragment integration
- ssDNA gap repair
Explain ssDNA gap repair
ssDNA gap repair:
1) ssDNA gap
2) RecA loaded by RecFOR
3) HJ formation
4) RuvAB loads onto HJ - direction of HJ migration along the strands
5) DNA replication
6) HJ resolution and D loop cleavage
7) DNA ligation
Explain ds break repair
dsDNA break repair:
1) dsDNA break
2) RecA loaded by RecBCD
3) HJ formation
4) RuvAB loads onto HJ - direction of HJ migration along the strands
5) DNA replication
6) HJ resolution and strand cleavage
7) Strand crossover
8) DNA ligation
What are the differences between RecFOR and RecBCD mechanisms to load RecA?
RecFOR (ss) - single RecA nucleoprotein filament added to non-broken strand
RecBCD (ds) - two RecA nucleoprotein filaments added to each 3’ overhang end of broken strand
Explain in detail the steps how RecBCD works to load RecA to repair dsDNA breaks
dsDNA break RecA loading:
1) RecBCD degrades DNA until Chi site
2) RecBCD recognises Chi site - only degrades 5’ end, so 3’ end overhang
3) RecBCD changes its activity
4) RecBCD loads RecA onto 3’ overhang -> RecA nucleoprotein filament
Explain the roles of RecBCD strutures pre-Chi / post-Chi site recognition
RecBCD - 3 subunits: B, C, D
1) Pre-Chi site recogntion
- RecB: helicase (unwinds) + nuclease (cuts 3’, 5’)
- RecC: Chi-scanning site
- RecD: helicase (unwinds)
2) Chi site recognition -> RecBCD conformational change
3) Post-Chi site recognition
- RecB: helicase (unwinds) + nuclease (cuts 5’)
- RecC: ??
- RecD: no activity
What are Chi sites?
Chi sites - multiple GCTGGTGG sequences in bacterial genome that regulate homologous recombination
RecBCD function overview
What are the roles of RecA nucleoprotein filament?
Roles of RecA nucleoprotein filament:
- homology search
- synapsis
- strand exchange
Does RecA coat ss or ds?
RecA coats ss but in ds repair sometimes goes over a bit into ds region
Explin how RecA nucleoprotein filament is formed
on ssDNA SSBPs - RecA recruited by RecFOR/RecBCD - nucleation - extension - some extension into ds region
Explain the process of homology search RecA
RecA homology search:
1) RecA searches by sliding along dsDNA strand
2) When homologous site found - alignment - base pairing
-> DNA synthesis initiated using homologous strand as a template
What is a D-loop?
Displacement loop (D-loop) - a crossing of ssDNA strands that displaces ssDNA from their original position
Explain DNA strand exchange after homology was found and stabilised
After homologous site found and pairing stabilised:
1) 3-stranded pairing intermediate of RecA covered broken DNA and homologous found strand
2) One strand released from the intermediate - exit site at branch point
3) Branch migration - where HJ will be formed
4) One ssDNA strand left behind
Explain strand invasion and Holliday junction formation
1) RecA slides along chromsome to find homology - base pairing
2) 3-stranded pairing - one ssDNA strand removed
3) RecA invades the homologous region - forms D-loop
4) DNA synthesis using homologous strand as template
5) HJ formation between the two strands
6) HJ resolving by RuvABC complex (HJ recognition by RuvA, RuvB migrates HJs in an ATP dependent process, RuvC cleaves HJs to separate the strands)
7) Ligation
8) Repair complete
Overview of full ssDNA, dsDNA break repair by RecFOR and RecBCD
What is the evidence for Holliday junctions?
Bacterial plasmid - cut with restriction enzymes - if recombined will be X form
Explain what is Holliday junction migration
HJs can move = branch migration
How can a Holliday junction be cleaved?
Horizontally: AB + A’B’
Vertically: AB + A’B’ or A’B + AB’
How to turn the chromosome for vertical HJ cleavage diagram?
What is the RuvABC binding to a Holliday junction, what is the role of RuvABC?
RuvABC role:
- RuvA - holds HJ in square planar configuration
- RuvB - directs branch migration of HJ
