Vertebrate NHEJ Flashcards
(23 cards)
1
Q
What is NHEJ? (4)
A
- Non-homologous end joining (template-free end joining) so can be mutagenic
- Involves proteins such as Ku70-80, DNA-PKcs, Artemis
- Backup pathway is microhomology-mediated end joining
- NHEJ is predominantly G1 phase, HR is S/G2 because template required
2
Q
How does NHEJ differ from HR? (3)
A
- NHEJ only does very short resection with tiny regions of homology
- Template-independent so can do any time
- Will probably lose a bit of sequence so mutagenic as by the time the repair machinery reaches the break site there is likely to be damage meaning the ends are incompatible and can’t just be re-ligated
3
Q
How does NHEJ differ in E. coli and vertebrates? (3)
A
- Bacteria: 2 component minimal system involving a protein that recognises the DNA ends and a ligase
- Mostly convergent evolution between species
- Vertebrate pathway much more complex
4
Q
What is Ku70-80? (2)
A
- Ku70-80 heterodimer made up of 70kD protein and 86kD protein
- Homology between them throughout several conserved domains but differences at the C termini
5
Q
How does Ku70-80 work? (5)
A
- Binds to ends of DNA to protect from degradation and hold them together
- Fits into minor and major groove contours (wraps around)
- Can even bind DNA associated with nucleosomes
- Forms a ring around DNA so can only get on when there is a break
- Acts as a ‘tool belt’ as other NHEJ proteins interact with Ku
6
Q
How is Ku linked to telomeres? (3)
A
- Chromosome ends look like DSBs so Ku binds to capped telomeres and protects from recombination and degradation
- This is important for silencing gene expression in telomeric regions
- But promotes telomere fusion at dysfunctional telomeres lacking the shelterin complex (cap) as can initiate NHEJ
7
Q
Which complex is Ku part of in eukaryotes?
A
Forms the DNA-PK complex with DNA-PKcs
8
Q
How is DNA-PKcs recruited to DNA? (2)
A
- Ku binds to DNA and recruits DNA-PKcs (catalytic subunit/p350) within a few seconds of the break forming
- DNA-PKcs is a DNA-dependent protein kinase (no kinase activity without Ku and DNA) and is controlled by phosphorylation at 2 different types of sites (40 sites on the protein in total) autophosphorylation or transphosphorylation
9
Q
What is autophosphorylation of DNA-PKcs?
A
Required for end ligation because it moves DNA-PK out the way so it’s not blocking DNA ends any more
10
Q
What is transphosphorylation of DNA-PKcs? (2)
A
- Generally mediated by ATM kinase
- Needed for recruitment of Artemis and therefore for end processing
11
Q
What is the synaptic complex?
A
Holds the broken DNA ends together
12
Q
How does synapsis of the DNA ends occur? (3)
A
- Long-range complex with ends tethered but far apart, requires DNA-PKcs to be present but not its catalytic activity
- Then ends are brought closer together which requires catalytic activity, where ligation will occur
- Ligases and other factors from downstream are also needed for the close-range complex to form
13
Q
Which ligases are involved in NHEJ? (2)
A
- DNA ligase IV with cofactor XRCC4 (X-ray repair cross-complementing protein 4)
- XLF (important for blunt-end ligation)
14
Q
How does blunt end ligation happen? (2)
A
- Neat blunt ended ligation/complementary overhangs can be simply ligated back together with DNA ligase IV+XRCC4/XLF
- Big complex with ligases and upstream factors at the DNA ends
15
Q
How does end-processing occur in NHEJ? (3)
A
- DNA ends may have been modified/damaged by nucleolytic degradation so need processing before ligation requiring nucleases/polymerases
- In particular, removal of blocking end groups by PNKP
- In humans Polμ and Polλ add nucleotides at the DNA junction in a template-dependent or independent manner
16
Q
What is PNKP? (2)
A
- Polynucleotide kinase/phosphatase
- Restores ligatable 3’ hydroxyl and 5’ phosphate groups by removing groups blocking the DNA ends
17
Q
What is the role of Artemis? (5)
A
- Nuclease recruited to DNA ends by Ku to help clean them up for ligation
- Activated by DNA-PKcs kinase
- Cuts many DNA substrates at the boundaries between ss and dsDNA
- Has intrinsic 5’ exonuclease activity on ssDNA
- Has endonuclease activity on 5’ and 3’ overhangs and on DNA hairpins when in complex with DNA-PKcs
18
Q
What is the iterative model of NHEJ? (2)
A
- Ends can be processed/repaired in multiple different ways and combinations of ways meaning the outcome is very unpredictable
- Move backwards and forwards between long and short-range complexes to ensure ligation is done as soon as possible to avoid over-processing of the DNA ends
19
Q
How is a decision made between repair pathways for DSBs?
A
- Resection of the ends is the essential decision point as HR generates a long ss region for strand invasion which is too big for NHEJ to deal with
- Mre11 (MRN complex) resection channels the DSB into the HR pathway
20
Q
What is alternative NHEJ? (4)
A
- Also called microhomology-mediated end joining
- Used when ends have been processed with Mre11 but then can’t go through with HR
- Extensive resection of DNA ends use use of small regions of homology uncovered to pair DNA strands
- More mutagenic than classical NHEJ (backup) as quite a lot of DNA can be lost
21
Q
How does alternative NHEJ work? (5)
A
- Break is bound by PARP rather than Ku
- Resection to reveal microhomology
- Flap removal
- Filling in by error-prone Polθ
- Ligation
22
Q
How does the cell cycle impact DNA repair pathway choice? (4)
A
- HR favoured at S and G2 phase
- NHEJ favoured at G1 phase
- Bacterial NHEJ is nonessential when cells are growing healthily because replication is constant to a duplicate genome is present to provide a template for HR
- During mitosis HR and NHEJ are both repressed and microhomology-directed end joining predominates
23
Q
How are kinases involved in repair control? (4)
A
- Complex interactions of different kinases including cell cycle kinases
- ATM (master regulator of DDR) blocks NHEJ
- ATM and ATR are in the same kinase family as DNA-PKcs
- DNA-PKcs also negatively regulates ATM via phosphorylation
