Flashcards in DSB 2 Deck (47)
What are the 2 major DSB repair pathways?
What is non homologous end joining?
- Ligating broken ends of DNA
- Doesn't require homology
- Error prone process
At what stage in the cell cycle does NHEJ function?
G0 and G1 phase of the cell cycle
Describe the NHEJ pathway
1. Recognition and end protection using Ku70/80 and DNA-PKcs
2. DSB end processing using Ku70/80, DNA-PKcs, PNK, artemis, Mre11, polymerase
3. DSB ligation using XLF/XRCC4 and LigIV
What is DNA PK how does DNA PK get activates
- DNA PK is a protein kinase
- It gets activated by binding to KU complex, KU complex doesn't form unless there is a DSB
What is the KU complex?
- A heterodimer that binds to ds DNA ends without specificity (400,000 per cell)
What does the KU complex do?
Protects DNA ends from nucleolytic degradation and other aberrant processing
What is DNA PKcs?
- It is a PIKK
- Very large protein- 469 KDa
Name 2 other members of the PIKK family
ATM and ATR
What is CDNA?
How big is CDNA?
Describe the structure of DNA PKcs
- Has lots of alpha helical HEAT motifs to allow bending and folding into hollow crystal structure
What is the most prevalent DSB repair?
Phosphorylation by histone H2AX
What is the role of histones?
To allow DNA to be compact in cells
What makes up a nucleosome?
H3, H4, H2A and H2B - 2 of each subunit (histone octamer)
How many base pairs does each histone octamer coat?
Name a variant of H2A
What are histones phosphorylated on?
N terminal tail
Name a substrate of DNA-PKcs
How can we study DSB repair in vivo?
1. Stain DNA with DAPI (fluorescent dye)
2. Irradiated for 15 -30 minutes = red spots.
Red spots indicate antibodies detecting phosphorylated histones (DSB)
3. After 180 minutes, less red spots visible because DSBs have been repaired
How is the release of DNA PKcs from KU regulated?
DNA PK has 3 clusters of auto-phosphorylation sites
What is artemis?
A nuclease involved in NHEJ using VDJ recombination
What does DNA PK activate?
artemis, XRCC4 and DNA ligase 4
What is processing actually for?
If DNA ends aren't perfectly compatible, then we need processing
What are the roles of artemis?
- Processes hairpins ends during VDJ recombination
- End trimming of IR induced DSBs
- heterochromatic DSB repair
What are the roles of PNKP?
- It has 3' phosphatase activity
- 5' kinase activity
- this allows removal of non ligatable end groups
What is MRN complex made up of?
- Mre11 (nuclease)
What is the role of Mre11?
- For DNA end binding
- 3' to 5' exonuclease
What is the role of DNA polymerase
Fill any gaps after ligation at the end of NHEJ