Homologous Recombination

Question 1

Genetic recombination

Answer 1

Recombination is a variety or processes that involve breakage and reunion of polynucleotides. It increases genetic diversity

Question 2

General or Homologous recombination (HR)

Answer 2

Occurs between two homologous DNA duplexes – Extensive sequence homology
Overall organization of DNA doesn’t change
Within any part of the chromosome

Question 3

Specialized recombination

Answer 3

Occurs only between specific short sites of DNA
- Short sequences of homology
Often makes changes in the organization of DNA

Question 4

Gene conversion

Answer 4

the process by which one DNA sequence is replaced by a homologous sequence such that they become identical. It happens after the resolution of the Holliday junctions.
After the mismatch repair system repairs one of the strands

Question 5

Homologous recombination: Steps

Answer 5

1. DSB initiates recombination
2. Exonuclease (5’ ->3’ activity) 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 (energetically neutral).
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 junctions is a critical step in HR

Question 6

The RecBCD pathway in E. coli

Answer 6

Chi sites are hotspots that stimulate recombination.
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. At same time RecB degrades one strand with (3’->5’)
exonuclease activity (∼1kb/s) until the Chi site. Complex stops at Chi site (there is a 5’ overhang)
4. RecB switches to endonuclease activity and cuts the other strand near the Chi site (leaves a 3’ overhang)
5. RecD detaches, it produces a conformational change that abolishes RecB nuclease activity. Only helicase
6. 3’ overhang is used as substrate for homologous recombination to performsingle-strand invasion, mediated by protein RecA

Question 7

The RecA protein in bacteria

Answer 7

a DNA-dependent ATPase.
- Activates the SOS response -> Stimulates protease activity
- Promotes single strand invasion (base pairing between a singles strand DNA and its complement in a duplex molecule)

Question 8

The RecFOR system in E. col

Answer 8

HR pathway that repairs single strand (SS) gaps, it requires three proteins: RecF, RecO and RecR.
But if RecBCD is abolished, RecFOR can also repair DSBs and take over from the RecBCD system
Similar steps to RecBCD, it also requires RecA for strand invasion and DNA exchange

Question 9

RuvABC proteins

Answer 9

• RuvAB complex is a molecular rotor. Rotate the DNA helices to allow branch migration (10-20 bp/second). Uses ATP as energy source
• RuvA (tetramer) recognizes the structure of the Holliday junction. Joins to the four DNA strands
• RuvB is a hexameric helicase with ATPase activity. It has a ring shape and binds around two DNA duplexes
• RuvC is a resolvase (endonuclease that recognizes Holliday junctions). Cuts in the tetranucleotide hotspot ATTG, that directs resolution.

Question 10

branch migration

Answer 10

A breakage and reformation of identical base pairs

Question 11

Chi sites

Answer 11

Hotspots that stimulate recombination. Sequence of 8 nucleotides (5’-GCTGGTGG-3’) every 5 to 10 kb. Recombination takes place around these chi sites.