Lecture 8 Flashcards
What happens if damaged DNA replicated?
- Mismatch base pair - Usual replication machinery - mutation
- DNA pols can’t replicate damaged base containing DNA
- Specialised translesion synthesis DNA pols replicate some DNA with damaged template
Are TLS DNA polymerases more flexible than replicative DNA pols?
Yes - Allows bypassing of problem DNA - Synthesis continued
What are translesion synthesis DNA polymerases formed from?
UmuDC and Beta clamp
What does DNA damage response induce?
- Increased DNA repair proteins
- Delay cell cycle
- Apoptosis
DNA damage response
ssDNA and DNA DSBs recognised by damage sensor proteins
- SOS response
- RecA is a multifunctional DNA binding protein
- LexA is a repressor - prevents SOS gene transcription by binding operator
- LexA binds as dimer at sequences similar to TACTG(TA)5CAGTA
- RecA binds and forms filament on ssDNA - cleaves LexA
Cleaved LexA can’t bind DNA - SOS genes aren’t transcribed
Process of DNA damage response
- RecA binds and forms filament on ssDNA - cleaves LexA
- Cleaved LexA can’t bind DNA - SOS genes aren’t transcribed
- Genes under SOS regulation encode
DNA repair proteins e.g. UvrA, B, D, RecA, RuvA, RuvB - Also encodes TLS pols (DinB, UmuC)
- SulA inhibits cell division
What do induced repair proteins do?
- Repair DNA damage
- ssDNA decreases
- RecA assembly and LexA cleavage reduced
- DinI protein mimics DNA
- LexA binds SOS boxes, SOS genes repressed
DNA damage response in eukaryotes
- DNA damage sensors recognise damaged DNA
- Damage sensors recognise different transducer kinase to damage site - ATR, ATM, DNA-PKcs
- Activate downstream proteins that recruit effector proteins when phosphorylated
RPA senses ssDNA at stalled replication forks
RPA binds ssDNA
RPA removed when lagging strand replicates
DNA damage causes DNA pol to stall - helicase unwinds
- RPA remains bound to ssDNA
How does RPA accumulation cause ATR activation
RPA recruits ATR via adaptor (ATRIP) - binds both
RPA recruits sliding clamp:clamp loader complex
- Damage specific sliding clamp is 9-1-1 (RAD9-RAD1-HUS1)
9-1-1 recruits TOPBP1 - activates ATR
What does activated ATR do?
Phosphorylates checkpoint kinases, RPA etc
Modulates cell cycle control, replication fork stabilization, Replication origin control
Stressed replication forks recruit polymerases
- ATRIP, ATR, and RPA bound to ssDNA
- Complex recruited that monoubiquinates PCNA
- Replicative pols have less affinity for PCNA so they disasssociate
- TLS pols have affinity for PCNA
- TLS pols resume DNA synthesis
What senses DSBs
MRN - Mre11, Rad50, Nbs1
MRN interacts with DNA at break
- MRN recruits ATM
- ATM exists in cell as inactive dimer
- ATM with MRN/DSB complex causes autophosphorylation and ATM activation
Name 2 alternative pathways in DSB repair
Non-homologous end joining - rejoins ends
Homology-directed repair - Uses homologous DNA as template - Late S-phase/G2 - sister chromatid available as template
NHEJ pathway
- Readily available in G1
- Nuclease digestion removes nucleotides before ligation OR resection exposes single-stranded regions
- NHEJ occurs by simple ligation of ends but end processing is usual
- Sequence loss - NHEJ is mutagenic
How do ATM and ATR mediate cell cycle arrest?
Phosphorylate checkpoint kinases CHK1 and CHK2
What do CHK1 and CHK2 phosphorylate?
CDC25A and CDC25C phosphatases - can’t remove inhibitory phosphates from CDK
- Can’t dephosphorylate CDK2-Cyclin E - blocks G1 to S transition
- Can’t dephosphorylate CDK1-CyclinB - Blocks G2 to M transition
p53 halts cell cycle
- p53 phosphorylated by ATR/ATM and CHK2
- Phosphorylated p53 forms tetramer - binds DNA
- p53 stimulates p21 expression
- p21 inhibits CDK 2-cyclin E kinase - blocks G1/S transition
