April 8 and 10 Flashcards
(22 cards)
What are the 3 PIKK of interest?
DNA-PKcs, ATM, ATR
PIKK catalytic domain is most similar to ?
PI3K
Heat repeat region looks like?
It is helix-loop l-helix …. Forms alpha solenoid
What is the most cytotoxic from of DNA damage?
When have DSB and other damage within 1 helical turn
Direct and indirect effects of ionizing radiation on DNA?
Direct? dnA
Indirect is water hydroxyl radicals
There are 2 major pathways of repair for IR-induced DNA DSBs. What are they, in what cell cycle stages are they active?
We have Non-homologous end joining (NHEJ) in G0,G1, S, G2, just not M phase. Fast and error prone
Then Hmologous recombintion repair (HRR)
It is S and G2, when have 2 pairs of chromatin
Slower and more accurate
Out of NHEJ and HRR, what is more common, issues?
It is NHEJ, This i serror prone. Does 80% of DSB repair
Steps in NHEJ?
- Ku70/80 binds to ends of ssDNA. 1 dimer per side
2.CTD of Ku is long tail, it interacts with DNA PKCcs
3.Have FAT, Kinase, and FATC domains as crown, the N terminal HEAT repeats encircle DNA - DNA-PK pushes Ku out of the way
- Autophosphorlation of ABCDE sites on DNA-PKcs, then released from Ku =large conformational change, DNA ends now close together
- XRCC4 saffold holds DNA ligase 4, which will do the work and ligate ends together
How is DNA-Pk and Ku interaction prevented?
DNA-PK autophosphorylates on SQ/TQ sites, so can’t interact with Ku, needs Mg-ATP. If we mutate these serine and thr, then they can’t be PO4’d and so even with Mg-ATP, don’t get dissociation
What is XRVCC4 as part of complex ith DNA ligase 4?
It is flexible coil region, ordered head region. It interacts wth BRCT domains of DNA ligase 4. Theis measn that Ligase 4 is always attached to XRCC4
What is order of what forms complex for NHEJ?
First Ku, then DNA-PKcs, then XLF/XRCC4
Steps for HRR?
Have MRN/CtIP bind at DSB
2. Short range resection, remove bases
3. Then Exonuclaes Exo1 and DNA2 remove more DNA, get ssDNA. Get long 5’ Overhangs
4. RPA binds to ssDNA
5. BRCA displaces RPA, allows Rad51 to bind to ssDNA
6. Start getting the template strand doing repair thing (HRR)
What does ATM stand for?
Ataxia Telangiestasia Mutated
Steps for ATM pathway (Rough)?
- MRN first binds near DSB
- ATM is dimer but then binds to DSB as monomer via Nbs1 on the MRN complex, it autophosphorylates
- ATM then PO4’s p53, Ch2, H2AX …..
What 3 proteins in MRN complex? How hold onto DNA? What also binds to Nbs1?
Mre11 (nuclease), Rad50, Nbs.
Have RAD50 with coiled arms to hook around DNA, then other stuff is the crown
Nbs! has CtIP on N terminus binding, then ATM on the C terminus
ATM activated will PO4 p53 and Chk2. Then what other step will fully activate p53?
Active Chk2 will then add second PO4 to p53 to fully activate it
Once active p53 leads to more p21 produced, What does Cdk2 do in what part of cell cycle? Inhibited by what?
It regulates G1 to S phase transition. It is inhibited by P21. more P21 means that more Cdk2 inhibition so better checkpoint from G1 to S
What is H2AX?
It is variant of H2A, it can be PO4’d on C-terminal tail by ATM, DNA-PK and ATR, so detection of it measures where cell damage occurs and how much as there is so much H2AX present, good signal
How does histone subunit H2AX help DNA repair
Once PO4’d by DNA-PK, ATM, or ATR, it has MDC1 (mediator of cell checkpoints 1) bind to it, and it too gets PO4d by same PIKKs. This leads to signal amplification, get more repair as more MDC1 binding with BRCT domains and therefore more MRN/ATM recruited
ATR Activation steps?
DSB in G2 phase
2. MRN/CtIP resection of DNA
3. Exo1 and DNA2 extend the 5’ overhang
4. RPA recruited, ATR-ATRIP bind and get activated
5. Again BRCA2 moves RPA so Rad51 can bind
6.
ATR PO4’s Chk1
ATM PO4s ?
ATR= S phase, binds to RPA. Then Chk1 PO4’d, which PO4’s CDC25 phosphatase and inhibits it, so now Phosphates are left on CDK2, it remains inactive
Chk2, get active p53, so more p21 that inhibits CDK2
