Chromosome biology lecture 11

Question 1

Genome size + replication rate

Answer 1

• E coli = 4.7Mbp to replicate in 20-30 mins
• Human = 3000Mbp, ↑ DNA
• Issues w/ DNA replication = big genome, only want to replicate once, needs to be accurate, all links btw 2 strands need to be removed,

Question 2

MMR

Answer 2

• Repairs base-base mismatches
• Replication slippage = type of error
• x detect a chemical change, hard to detect
• Solution = identify newly synthesised strand

Question 3

Discriminating daughter + parental strand

Prokaryotes

Answer 3

• Identification of mismatches = methylation status of new DNA at GATC
• After replication, window where parent = methyl, daughter x (hemi-methylated)

Question 4

Methyl-directed MMR

Answer 4

• At methylated GATC, MutH aspic
• MutS binds mismatch + communicates to MutH via MutL
• Activates endonuclease activity of MutH, cleaves daughter
• Nick unwound by UvrD
• DNA synthesis

Question 5

How is MutH activated

Answer 5

• Different models
  1. translocation model = extrusion of DNA through MutS dimer pulls MutH towards it
  2. Sliding clamp where MutS moves to MutH
  3. Spooling at 1 side of MutS dimer to reel MutH close

Question 6

Discriminating btw daughter + parental strand

Eukaryotes

Answer 6

• Resection = issue for lagging strand as x know directionality
• Achieved w/ PCNA (maintained on lagging strand)
• MutS, L translocate, if meet PCNA in good direct, resect towards mismatch

Question 7

Translesion synthesis

Answer 7

• Damaged lesion encountered by replication forks stalls
• Could use HR to restart replication, then repair w/ NEJ
• Replicate past lesion w/ Pol w/ ↓ fidelity so can replicate past lesions

Question 8

Polymerase switch

Answer 8

• Rad6/MMS2 + Ube13 = ubiquitin conjugating E
• Rad18/5 = ubiquitin ligase
• When a replication fork encounters damage, PCNA is mono-ubiq
• PCNA becomes poly ubiq, has ↑ affinity for PolN, Pol, allowing translation synthesis
• Synthesises past damage

Question 9

DNA interstrand crosslink repair

Answer 9

• Covalent bond btw 2 bases on opposite strand (x use helicase or recombination)
• Combination of pathways
• e.g. = NER to cut out damage, HR to restart replication

Prokaryotes
cross-link w/ UvrABC → unhook lesion to give gap + ssDNA → RecA to invade if homologous chromosome

Eukaryotes
FA pathway, FA core recruits key FA protein → assembly of HR/nucleases that process ICL → unhook ICL one lower strand of DNA, fill in w/ DNA replication + bypass w/ translesion synthesis

Question 10

ICL defective repair

Answer 10

• Can try using another pathway
• Experiment w/ WT or knock down FRANCD2
• If compromised NHEJ can rescue w/ FA