Chromosome biology lecture 10

Question 1

Sources of DSB

Answer 1

• O2 radicals
• Ionising radiation
• DNA cleaving agents e.g. bleomycin
• If replication fork encounters SSB → replication run-off
• 9 per day, most cytotoxic (illegitimate joining/breakdown by nucleases)

Question 2

Pros/Cons of DSB

Answer 2

GOOD
Generated under normal conditions e.g. meiotic recombination, V(D)J recombination

BAD
Crossing over → loss of heterozygosity, recomb. btw repetitive DNA → loss of genetic info, de-reg DSB repair → gross chromosomal rearrangement, aberrant ds break repair → loss of genome integrity

Question 3

HR in E coli

Answer 3

• Need homologous chromosome
• DS break w/ homologous sequence for repair, detect ds break, resect DNA ends → 3’ overhand, RecA facilitates strand invasion, synthesis using 3’OH as primer, holiday junction resolution
• Conserved in vertebrates
• Resolving
  1. Double Holliday junction cleaved w/ nuclease
  2. Migration of both Holliday junction together → catenene, resolved w/ topoisomerase
  3. SDSA = HJ migration that flips out ss structure making homologous region that can anneal, then fill in gap

Question 4

NHEJ overview

Answer 4

• Process of direct DNA end-end fusion that x use strand exchange or homologous DNA
• DSBs detected, bound by proteins that facilitate assembly of repair factors
• End of DSB = often ‘dirty’, need to process 3’P (can → loss of genetic info)

Question 5

VDJ recombination

Answer 5

• Variable region fused to joining region inaccurately to cause mutation to provide ↑ diversity

Question 6

Components of NHEJ pathway

Answer 6

• Ku70/80 heterodimer binds DNA
• Artemis = 5’-3’ exonuclease
• MRN = 3’-5’ exonuclease
• DNA ligase IV

Question 7

NHEJ pathway

Answer 7

• MRN + assoc. Ctip bind DNA damage in response to IR
• Mutation in Mre11 → genomic instability syndrome like ATLP
• MRX assoc transiently w/ regions proximal to DSB
• MRN polarity = wrong direction, only 100bp resected
• Use exo1 (5’-3’ exonuclease) + Sgs1-DNA2
• 3’ overhand facilitates Rad51 invasion

Question 8

HR vs NHEJ

Answer 8

• Regulation of DNA end resection = determinant
• HR x good in G1 as x have homologous chromosome
• CtIP needed to activate MRN/MRX
• CtIP phosph on Ser267 by Cdc28 which is needed for Clb/Cln activation