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DSB 2 Flashcards

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1
Q

What are the 2 major DSB repair pathways?

A
  1. NHEJ

2. HR

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2
Q

What is non homologous end joining?

A
  • Ligating broken ends of DNA
  • Doesn’t require homology
  • Error prone process
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3
Q

At what stage in the cell cycle does NHEJ function?

A

G0 and G1 phase of the cell cycle

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4
Q

Describe the NHEJ pathway

A
  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
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5
Q

What is DNA PK how does DNA PK get activates

A
  • DNA PK is a protein kinase

- It gets activated by binding to KU complex, KU complex doesn’t form unless there is a DSB

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6
Q

What is the KU complex?

A
  • A heterodimer that binds to ds DNA ends without specificity (400,000 per cell)
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7
Q

What does the KU complex do?

A

Protects DNA ends from nucleolytic degradation and other aberrant processing

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8
Q

What is DNA PKcs?

A
  • It is a PIKK

- Very large protein- 469 KDa

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9
Q

Name 2 other members of the PIKK family

A

ATM and ATR

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10
Q

What is CDNA?

A

DNA-PKcs

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11
Q

How big is CDNA?

A

14 KB

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12
Q

Describe the structure of DNA PKcs

A
  • Has lots of alpha helical HEAT motifs to allow bending and folding into hollow crystal structure
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13
Q

What is the most prevalent DSB repair?

A

Phosphorylation by histone H2AX

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14
Q

What is the role of histones?

A

To allow DNA to be compact in cells

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15
Q

What makes up a nucleosome?

A

H3, H4, H2A and H2B - 2 of each subunit (histone octamer)

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16
Q

How many base pairs does each histone octamer coat?

A

150

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17
Q

Name a variant of H2A

A

H2AX

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18
Q

What are histones phosphorylated on?

A

N terminal tail

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19
Q

Name a substrate of DNA-PKcs

20
Q

How can we study DSB repair in vivo?

Study These Flashcards
A
  1. Stain DNA with DAPI (fluorescent dye)
  2. Irradiated for 15 -30 minutes = red spots.

Red spots indicate antibodies detecting phosphorylated histones (DSB)

  1. After 180 minutes, less red spots visible because DSBs have been repaired
21
Q

How is the release of DNA PKcs from KU regulated?

Study These Flashcards
A

DNA PK has 3 clusters of auto-phosphorylation sites

22
Q

What is artemis?

Study These Flashcards
A

A nuclease involved in NHEJ using VDJ recombination

23
Q

What does DNA PK activate?

Study These Flashcards
A

artemis, XRCC4 and DNA ligase 4

24
Q

What is processing actually for?

Study These Flashcards
A

If DNA ends aren’t perfectly compatible, then we need processing

25
What are the roles of artemis?
- Processes hairpins ends during VDJ recombination - End trimming of IR induced DSBs - heterochromatic DSB repair - Nuclease
26
What are the roles of PNKP?
- It has 3' phosphatase activity - 5' kinase activity - this allows removal of non ligatable end groups
27
What is MRN complex made up of?
- Mre11 (nuclease) - Rad50 - Nbs1
28
What is the role of Mre11?
- For DNA end binding - Endonuclease - 3' to 5' exonuclease
29
What is the role of DNA polymerase
Fill any gaps after ligation at the end of NHEJ
30
What is the difference between endonuclease and exonuclease?
Endo - removes from within DNA molecule | Exo - removes from end of DNA
31
What is the ligation step of NHEJ?
XRCC4 and lig4 is recruited to DNA via DNA PK and forms a tight complex (XLF is a weaker binding partner)
32
What does DNA ligase 4 do in general?
Catalyses a covalent phosphodiester bond on a wide range of DNA end structures (VERY FLEXIBLE)
33
What XLF for?
For ligase 4 re-adenylation
34
What is NHEJ ligase complex?
DNA ligase 4 XRCC4 XLF
35
Why do lig4 and xrcc4 need to be together in the ligase complex?
To ensure ligase 4 is stable and functional
36
What are the phenotype of cell lines that are deficient in NHEJ?
- Defective in VDJ recombination and class switch recombo - Sensitive to ionising radiation and anything that induces DSBs - Some endogenous genomic instability - No UV sensitivity unless at replication fork
37
What do KU KO mice show?
- Small - SCID - Premature ageing
38
What do DNA PKcs defective mice show?
- SCID | - BUT grow at normal rate/size
39
What do lig4/XRCC4 KO mice show?
Embryonic lethal (neuronal apoptosis)
40
Which steps of NHEJ are essential/not essential?
- Ligation is essential for survival | - DSB recognition/protection is not essential, it just enhances the repair
41
What are the effects of defective ligase 4 and XLF?
Lig4/XLF syndrome: - Pancytopaenia - Delayed development - Facial features/microcephaly - Chromosome instability - Radio-sensitivity
42
What happens if there is a mutation in artemis?
RS-SCID - immunodeficiency - no neurological developmental defects
43
What is the role of ATM signalling?
- Role is DSB repair via modulation of heterochromatin structure - Heterochromatin is a barrier to DSB repair that ATM overcomes by recognizing formation of repaired breaks, halt cell cycle and promote recruitment of additional repair factors
44
What is the difference between euchromatin and hetero chromatin? (SLIDE ASIDE)
- Euchromatin is more accessible - Hetero chromatin is rarely used and does not need to be accessed (Both need to be replicated before division)
45
What is the difference between NHEJ and ATM signalling?
- NHEJ required for fast and slow repair | - ATM required only for slow repair. It is needed for 10-15% of DSBs.
46
Is NHEJ and error free mechanism?
- No, can be error prone, leading to chromosome translocations, deletions, mutations etc.
47
SIDE NOTES
- NHEJ deficient cells are highly radiosensitive - But doesn't mean they're not sensitive DNA damaging agents directly - Instead DSBs happen indirectly through replication pr UV exposure.

Genetics (9 decks)

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