Homologous Recombination

Question 1

Homologous recombination steps

Answer 1

1. DSB initiates recombination
2. Exonuclease (5’->3’) degradation of the 5’ ends. Creates 3’ overhangs
3. Single strand invasion of one 3’ ends into the homologous chromosome, it forms a D-loop structure
4. Heteroduplex is formed. The recombinant joint moves with Branch migration
5. Extension of the 3’end by DNA polymerase
6. Displaced D-loop pairs with the other strand and DNA polymerase fills the gap
7. The free 5’ end performs a second single strand invasion, creating a second recombinant joint
8. DNA ligase seals the nicks generating 4 complete DNA strands and 2 Holliday junctions
9. Resolution of the Holliday junction

Question 2

Possible outcomes for Homologous recombination:

Answer 2

1. Non crossover DNA (both cut in the same axis)
2. Crossover Recombinat DNA (Cut in different axis)

Question 3

RecBCD pathway in E.coli

Answer 3

Creates a 3’overhang as a substrate for Homologous Recombinatio
1. RecBCD complex binds to the DSB and travels along the DNA
2. RecB (3’-> 5’ helicase) and RecD (5’-> 3’ helicase) unwind the DNA
3. RecB degrades one strand with (3’ -> 5’) exonuclease activity until the Chi site. -> creates a 5’overhang
4. RecB switches to enduclease activity and cuts the other strand near the Chi site -> leaves 3’ overhang
5. RecD detaches, Recb no longer nuclease
6. 3’overhang used for homologus recombination to perform single-strand invasion mediated by protein recA

Question 4

RuvABC system:

Answer 4

Branch migration and resolution of Holliday junctions are catalyzed by the RuvABC system
1. RuvAB complex is a molecular rotor. Rotate the DNA helices to allow branch mirgation (ATP)
2. RuvA recognizes the structure of the Holliday junction. Joins the four DNA strands
3. RuvB helicase with ATPase activity
4. RuvC is a revolase. Cuts in the tetranucleotide hotspot ATTG,