L13-DSB repair

Question 1

how do we assay the DSB repair capacity of a cell?

Answer 1

using colony survival assays
- use 4 wells with the same amount of cells
- treat them with different IR conditions
-leave for 2 weeks
- stain the colonies and count the cells which survived
increased dose = decreased colonies = increased DSBs = increased cells killed
or
indirect immunofluorescence
- grow cells on slide and IR them
- fox with PFA and use triton X100 to permeabilise the cells
- incubate with 1 AB and wash
- incubate with 2 AB and wash
- incubate with DAPI to stain DNA
-add coverslip and use FACS microscope
= shows the number of foci formed which decrease overtime as repair occurs
use gammaH2AX as a marker

Question 2

describe the mechanism of DSB repair with NHEJ?

Answer 2

IR causes DSBs
ku70/80 binds to the DSBs with PAXX getting recruited and binding to the DNA ends
DNA-PKcs is recruited and is activated
artimas (DNA nuclease) trims a few nucleotide bases of the DSB ends

the 5’ and 3’ POLY-a tail and cap are removed by PNKP
ligation occurs using XRCC4 and XLF and recruit ligase IV to extend the DNA

Question 3

how do we assay NHEJ repair? (describe all 4 ways)

Answer 3

1. WB
   - IR cells the do SDS-PAGE and nitrocellulose membrane transfer
   - use Ponceou S
   1 and 2 AB
   HRP on 2 AB = DETECT LIGHT
2. IP kinase assay to measure kinase activity of DNA-PK
   -IR cells the lyse them
   -add anti-DNA-PKcs AB
   -add protein A/G beads binds to AB to isolate the DNA
   -isolate the AB-bead complex by centrifugation
   -wash and add substrate dsDNA and radio labelled ATP
   -incubate at 37C
   SDS PAGE
   stain gel dy and expo to radioactivity sensitive film
   =p increases with dsDNA breaks
3. immunofluorescence to assess the ability of NHEJ proteins to detect DSBs
   -grow cells and induce IR
   -tag with Brad
   use UVA laser and mark where DSBs are
   visualise
   =if GFP and GAMMAH2AX overlap = DNA damage detected
4. FACS, with reporter assay
   - use plasmid

Question 4

describe the process of alternative DNA end joining

Answer 4

IR causes DSB
in the absence of the NHEJ machinery (no ku70/80 therefore the MRN complex recognises it instead)
uncontrolled resection of DSBs occur via exo1
the MRN complex is recruited and a DNA synapse is formed by PolQ = which causes annealing and extension of the DNA strands
due to displacement, the DNA flaps are removed by Fen1
XRCC1 and PARP1/3 and ligase 3 are recruited to fully extend the DNA
lots of DNA is lost during this process

Question 5

how do we assay ALT-EJ?

Answer 5

using FACS based reporter assays

Question 6

describe the mechanism of homologous recombination?

Answer 6

IR induces DSB
detected by the MRN complex
endonuclease (CtIP) activity occurs
53bp1 COMPLEX limits resection and HR
RPA binds to the end strands
BRCA1/2 and PALB2 remove RPA and load on RAD51 INSTEAD
RAD51 promotes the adding of nuclear filaments around the SSDNA and invades the non damaged template
it binds to the identical sequence to its sequence and recruits DNA pol
it then uses this strand to repair the DSB
the 2 templates join and form a HJ
this HJ is repaired by either BLM/TOP3/RMI complex OR using exonuclease (SLX4)

Question 7

describe 3 ways to assay for HR

Answer 7

use imunoflourescne - to assess RAD51 ability to relocalise to DSBs
assess the sister chromatic exchange frequency -use parent karyotype
FACS based reporter assay - use GFP