Lecture 13: Biochemistry Of Homologous Recombination

Question 1

What are the 2 pathways to initiate DNA repair by homologous recombination?

Answer 1

RecFOR: single strand gaps
RecBCD: double strand breaks

Question 2

When do cells need to carry out homologous recombination?

Answer 2

DND double stranded break, dsDNA fragment integration, or DNA single strand gap

Question 3

What are the steps involved in single strand gap repair?

Answer 3

• recA is loaded on DNA by RecFOR
• recA catalyses finding homologous chromosome
• strands are exchanges to copy missing information
• form Holliday junctions which are coated by RuvA and RuvB
• RuvC cleaves strands
• leads to repair

Question 4

What are the steps involved in double strand break repair?

Answer 4

• ends of breaks are recognised by RecBCD
• RecB loads on DNA and forms single strand part of DNA at chi site which is then covered by RecA
• RecA can identify homologous regions on another copy of the chromosome
• invade and form branches that are migrated and resolved by RuvA and RuvB

Question 5

What roles does RecBCD have?

Answer 5

• DNA degradation
• recognises chi sites
• changes its biochemical activity and RecA is loaded by RecBCD

Question 6

How does action of RecBCD work?

Answer 6

• RecBCD recognises double strand end
• made of helicase B and D which allow opening of DNA and translocation occurs
• linked to activity of RecB subunit, which is nuclease, so degradation of 3’ and 5’ ends of DNA
• DNA degradation happens until chi site is encountered, stops degrading 3’ end, keeps degrading 5’ end
• loads RecA into single strand being formed
• triggers production of RecA protein filament used for homology search

Question 7

What are the roles of RecA

Answer 7

• catalyze homology search
• synapsis, which is when nucleoprotein filament connects with other homologous chromosome
• strand exchange

Question 8

How does RecA form nucleoprotein filament in single strand gap?

Answer 8

• single strand DNA in a gap is covered by SSB protein
• RecFOR loads few molecules of RecA through nucleation
• nucleoprotein filament through fast extension process
• extends DNA until end

Answer 9

• RecA nucleoprotein filament searches and invades homologous DNA sequence
• D-loop is formed
• triggers synthesis to repair missing DNA
• other end of the break is captured

Question 10

How does RecA find homologous sequence?

Answer 10

• catalyses short contacts between nucleoprotein filament and the chromosome in a random manner
• carries out intersegmental contact sampling to test 2 pieces at same time
• RecA can slide across DNA
• when homologous sequence is found, base pairing happens to form Dloop

Question 11

How are Holliday junctions recognised and resolved?

Answer 11

RESOLVED BY RUVABC COMPLEX
- each Holliday junction is recognised by RuvA which binds and keeps them open and mobile
- RuvA loads RuvB translocase which migrate HJs in an ATP dependent manner
- RuvC is an endonuclease, which cleaves the HJs
- nicks are ligated by replicative ligase