Module 2 Section 5

Question 1

What is homologous recombination (HR)

Answer 1

• recombination between 2 DNA mlc of similar sequence, occurs in all cells
• takes place during meiosis+mitosis in euk and during repair of DSB

Question 2

About eukaryotic meiosis

Answer 2

• pairs of homologous chromosomes in S phase
• homologous sister chromatid pairs align in prophase I
• 4 gametes produced (haploid)
• homologous chromosomes segregated in meiosis I, sister chromatids segregated in meiosis II
• cohesions (proteins) provide physical link between sister chromatids

Question 3

Roles of crossing over

Answer 3

1. creates physical link essential for proper chromosomal segregation
2. following segregation, sister chromatids (now daughter chromosomes) are no longer identical
   - parts of each set of chromosomes exchanged (generates genetic diversity)
   - visible is prophase I (chromosomes partially separate, still held together at chiasmata (joints))
   - many of same enzymes used in DNA repair +meiotic recombination, begins w DSBs

Question 4

Initiation of Meiotic recombination

Answer 4

1. following s phase homologous chromosomes brought together
   - as cell enters meiosis, DSB intentionally introduced at multiple locations (Spo11 catalyzes DSBs)
2. Spo11 uses active site Tyr as nucleophile in transesterification reaction
   - each subunit cleaves one strand
   - phosphodiester bond replaced by covalent 5’-phosphotyrosol linkage

Question 5

Transesterification reaction define

Answer 5

the process of exchanging the organic group of an ester with the organic group of an alcohol

Question 6

Processing of Spo-11-Mediated DSBs

Answer 6

1. Mre11-Rad50-Xrs2 complex binds to each Spo11 complex, cleaves DNA by several base pairs on 3’ side of Spo11
2. nuclease Sae2 degrades DNA more
   - other enzymes (Sgs1 helicase, Exo1/Dna2 nucleases) implicated in more extended degradation of 5’ ends and create long 3’ ss extensions
   - processed in mechanism similar to DSB repair

Question 7

Meiotic recombination completion

Answer 7

• completed by classic DSBR pathway
  1. ss regions bound by RPA (SSB protein), 2 RecA-class recombinases (Dmc1 and Rad51) leaded onto 3’ extensions on either side of DSB
  2. site is set up for recombination
  3. stable DNA joint formed by intertwining ssDNA with its compliment from homologous target
• polymerase + ligase complete the repair

Question 8

Crossover vs. non-crossover events

Answer 8

• cleaving crossover strands leads to completion of replication + segregation of 2 monomeric (mlc combines w same type) chromosomes in daughter cells
• cleaving the template strands creates chimeric (fusion of 2 kinds of mlc) chromosomes

Question 9

Catalysis of Holliday intermediate resolution

Answer 9

• resolved by nicking a strand in each duplex followed by ligation
• facilitated by RuvAB complex (up to 2 RuvA protein tetramers bind to Holliday intermediate and form complex with 2 RuvB hexamers

Question 10

RuvA protein

Answer 10

-Holliday junction specific DNA binding protein, recognizes the structure of the DNA junction

Question 11

RuvB protein

Answer 11

• donut shaped hexamer
• surrounds 2 of the 4 arms of Holliday intermediate
• is a DNA translocase
• DNS propelled outwards through hole in RuvB, away from junction
• rxn coupled to ATP hydrolysis
• results in rapid movement of Holliday intermediate

Question 12

RuvC protein

Answer 12

• RuvAB complex moved Holliday intermediate away from area of damaged DNA, it recruits RuvC
• is a Holliday intermediate resolvase
• replaces one of the RuvA tetramers at junction, cleaves strands in opposing arms of Holliday intermediate, nicks 2 strands with the same polarity (ie. same direction)
• nicks sealed into different arrangement by ligase