L11-Recognising DNA damage

Question 1

name 3 ways how DNA damage can be induced?

Answer 1

IR, X rays and chemotherapeutic drugs

Question 2

which cellular process/mechanism repairs DSBs and ICL?

Answer 2

HR AND NHEJ

Question 3

name 2 protein complexes which detect DNA DSBs?

Answer 3

Ku70/80 hetereodimer

MRN complex

Question 4

describe the process of the Ku complex recognising DNA DSBs?

Answer 4

1. the Ku70/80 heterodimer binds to the DNA DSBs at high affinity and sides it onto the DNA
2. to prevent DNA strands from separating the ku complexes have a protein, PAXX(which is bound to the ku complex via Ku binding domain (KBD)) which get dimerised and hold ku bound and stabile
3. ku then recruits DNA-PKcs (catalytic subunit) which transphosphorylate themselves and get activated
4. DNA-PKcs alsophosphotylates H2AX into gammaH2AX
5. gammaH2AX acts as a marker for DSBs

Question 5

describe the 2 lab techniques used to analyse DNA DSBs repair?

Answer 5

1. using fluorescence/fluorochrome microscopy to localise the protein of interest
   - transfect cells with a plasmid that encodes a DNA damage repair protein (e.g Ku) and tag it with a fluorescent marker such as GFP
   - then grow the cells in a culture dish and incubate with Brad (a nucleotide which is incorporated into the DNA )
   - expose the DNA to UVA laser which creates DSBs
   - create a stripe across the nucleus where the DSB is
   - look at the cells using RT-microscopy (a time course) to see Ku requirement
2. using antibodies to the specific protein of interest
   - grow the cells on a culture dish an treat them with either IR/UVA laser = to induce DNA damage
   - fix cells with PFA
   - permeabilise using triton-x100 =a allows ABs to enter cell
   - incubate with an AB to a protein of interest (e.g RAD51/KU)
   - this primary AB will bind to the protein of interest and then add a secondary AB which recognises the 1st with fluorochrome bound to it
   - stain with DAPI = visualise cells with fluorescent microcopy

Question 6

name and state how many of the components that make up the MRN complex?

Answer 6

• Mre11 (2X)
  -RAD51(2X)
  NBS1 1/1.5X)

Question 7

describe the process of how the MRN complex recognising DNA DSBs?

Answer 7

1. MRE11 and RAD50 dimers bind to each end of the DSBs
2. NBS1 binds to MRe11 via the MBD
3. NBS1 then recruits ATM via the AIM which allows the 2 BSB ends to be held together and bring together 2 ATM molecules
4. The ATM molecules then transphosphorylate each other and get activated
5. this then phosphorylates and activates the H2AX molecules forming gammaH2AX
6. GammaH2AX then forms a platform to recruit MDC1 (a phosphobinding protein) and also with FHA and BRCT1 domains on the NBS1 chains
7. NBS1 also binds to polyADP-ribose chains through PARP1 which stabilises the MRN complex at DNA damaged sites
8. this maintains NBS1 binding to chromatin via H2AX

Question 8

how does the cell decide to use Ku or MRN-dependent repair?

Answer 8

depending on the activity levels of CDKs

• during s phase, DNA damage is repaired by the MRN complex
• ku is not recruited during s phase due to ATM inhibiting it

Question 9

which repair mechanism repairs euchromatin or heterochromatin?

Answer 9

Ku = euchromatin DSBs
MRN = heterochromatin DSBs

Question 10

what is CtIP and describe its function in DNA repair?

Answer 10

CtIP is a protein that binds to the MRN complex to repair DNA via HR
Its phosphorylated by Cdk2
it also has nucleolytic activity