L12-DNA chechpoints

Question 1

what proteins controls the G1/S checkpoint

Answer 1

ATM/p53

Question 2

describe how DNA damage activates the G1 phase checkpoint

Answer 2

DSBs occur and causes recruitment of H2AX
H2AX gets phosphorylated into gammaH2AX via ATM (MRN complex )
ATM phopshorylates p53 and chk2
ch2 then phosphorylates cdc25A which removes phosphate groups from sdks
Cdc25A either undergoes degradation or phosphorylated cdk2 to bind to cyclin E
prevents S phase entry
or
p53 transactivated p21 which binds to the cdk2/cyclin E complex and inhibits S phase entry

Question 3

describe the 2 assay used investigate how cells activate the G1/S checkpoint?

Answer 3

1. using western blotting
   - IR the cells and lyse them
   - run them down a SDS-PAGE and transfer to nitrocellulose membrane
   - use a ponceau S stain to check if all transfer has been successful
   - wash of ponceau stain
   use 1 AB to detect POI (IE p53)
   then wash and add 2 AB coupled with HRP
   incubate with ECL and HRP turns it into light
   bar shown on blot
2. using FACS
   Incubate the IR cells with BrdU, its only incorporated in S phase
   trypsinase and fix with ethanol
   wash and incubate with HCL to allow DNA to become accessible
   use ABs to recognise Brad and use 2 AB with fluorophore
   incubate with PI and RNAse
   use FACS to visualise results

Question 4

which proteins control the intra-S phase checkpoint

Answer 4

ATM/ATR

Question 5

describe how DNA damage activates the S phase checkpoint

Answer 5

DSBs occur and gamma H2AX gets phosphorylated by ATM via the MRN complex
ATM then phosphorylates chk2 which phsoporylates cdc25A
cdc25A then is either targeted for degradation or inhibits cdk2/cyclin E/A
= halt at s phase

Question 6

describe the 2 assay used investigate how cells activate the inra-S checkpoint?

Answer 6

1. DNA fibre analysis
   - it assesses whether the firing of new organs is stopped after DNA damage
   INCUBATE cells with Cidu (thymine analogue) which is incorporated into the DNA
   wash off any excess and IR and incubate with IdU (another thymine analogue)
   put on glass slide and tip over so DNA runs
   DNA fibres made
2. WB with phosphospecifc ABs
   - it recognises proteins involved in regulating checkpoint e.g brca1 s1387-p
   check for SMC1 S966-P, SMC1, NBS1 and chk1 expression with ATR, Nbs1 should have decreased band after ATR

Question 7

which protein controls the G2/M checkpoint?

Answer 7

ATM/ATR

Question 8

describe how DNA damage activates the G2/M phase checkpoint

Answer 8

the DSBs are resected and ATM and ATR are phosphorylated
this causes chk2 and 1 to become phosphorylated and then phosphorylate cdc25B/C
causes cdk1 to be phosphorylated and bind to cyclin B
BLOCKS M PHASE ENTRY

Question 9

describe the 1 assay used investigate how cells activate the G2/M checkpoint?

Answer 9

1. use FACS
   - culture and IR the cells
   trypsinise them and fix with ethanol to allow AB to enter the nucleus
   incubate with1 AB that recognises phosphohistone H3 serine-10 ( a specific mitotic marker to test if the cells are in M phase)
   wash 1 AB off and add 2 AB coupled with GFP
   incubate with PI and RNAse
   = FACS analysis