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Flashcards in DSB 2 Deck (47)
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1
Q

What are the 2 major DSB repair pathways?

A
  1. NHEJ

2. HR

2
Q

What is non homologous end joining?

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

At what stage in the cell cycle does NHEJ function?

A

G0 and G1 phase of the cell cycle

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
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

6
Q

What is the KU complex?

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

What does the KU complex do?

A

Protects DNA ends from nucleolytic degradation and other aberrant processing

8
Q

What is DNA PKcs?

A
  • It is a PIKK

- Very large protein- 469 KDa

9
Q

Name 2 other members of the PIKK family

A

ATM and ATR

10
Q

What is CDNA?

A

DNA-PKcs

11
Q

How big is CDNA?

A

14 KB

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
13
Q

What is the most prevalent DSB repair?

A

Phosphorylation by histone H2AX

14
Q

What is the role of histones?

A

To allow DNA to be compact in cells

15
Q

What makes up a nucleosome?

A

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

16
Q

How many base pairs does each histone octamer coat?

A

150

17
Q

Name a variant of H2A

A

H2AX

18
Q

What are histones phosphorylated on?

A

N terminal tail

19
Q

Name a substrate of DNA-PKcs

A
  • H2A
20
Q

How can we study DSB repair in vivo?

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?

A

DNA PK has 3 clusters of auto-phosphorylation sites

22
Q

What is artemis?

A

A nuclease involved in NHEJ using VDJ recombination

23
Q

What does DNA PK activate?

A

artemis, XRCC4 and DNA ligase 4

24
Q

What is processing actually for?

A

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

25
Q

What are the roles of artemis?

A
  • Processes hairpins ends during VDJ recombination
  • End trimming of IR induced DSBs
  • heterochromatic DSB repair
  • Nuclease
26
Q

What are the roles of PNKP?

A
  • It has 3’ phosphatase activity
  • 5’ kinase activity
  • this allows removal of non ligatable end groups
27
Q

What is MRN complex made up of?

A
  • Mre11 (nuclease)
  • Rad50
  • Nbs1
28
Q

What is the role of Mre11?

A
  • For DNA end binding
  • Endonuclease
  • 3’ to 5’ exonuclease
29
Q

What is the role of DNA polymerase

A

Fill any gaps after ligation at the end of NHEJ

30
Q

What is the difference between endonuclease and exonuclease?

A

Endo - removes from within DNA molecule

Exo - removes from end of DNA

31
Q

What is the ligation step of NHEJ?

A

XRCC4 and lig4 is recruited to DNA via DNA PK and forms a tight complex

(XLF is a weaker binding partner)

32
Q

What does DNA ligase 4 do in general?

A

Catalyses a covalent phosphodiester bond on a wide range of DNA end structures (VERY FLEXIBLE)

33
Q

What XLF for?

A

For ligase 4 re-adenylation

34
Q

What is NHEJ ligase complex?

A

DNA ligase 4
XRCC4
XLF

35
Q

Why do lig4 and xrcc4 need to be together in the ligase complex?

A

To ensure ligase 4 is stable and functional

36
Q

What are the phenotype of cell lines that are deficient in NHEJ?

A
  • 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
Q

What do KU KO mice show?

A
  • Small
  • SCID
  • Premature ageing
38
Q

What do DNA PKcs defective mice show?

A
  • SCID

- BUT grow at normal rate/size

39
Q

What do lig4/XRCC4 KO mice show?

A

Embryonic lethal (neuronal apoptosis)

40
Q

Which steps of NHEJ are essential/not essential?

A
  • Ligation is essential for survival

- DSB recognition/protection is not essential, it just enhances the repair

41
Q

What are the effects of defective ligase 4 and XLF?

A

Lig4/XLF syndrome:

  • Pancytopaenia
  • Delayed development
  • Facial features/microcephaly
  • Chromosome instability
  • Radio-sensitivity
42
Q

What happens if there is a mutation in artemis?

A

RS-SCID

  • immunodeficiency
  • no neurological developmental defects
43
Q

What is the role of ATM signalling?

A
  • 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
Q

What is the difference between euchromatin and hetero chromatin?

(SLIDE ASIDE)

A
  • Euchromatin is more accessible
  • Hetero chromatin is rarely used and does not need to be accessed

(Both need to be replicated before division)

45
Q

What is the difference between NHEJ and ATM signalling?

A
  • NHEJ required for fast and slow repair

- ATM required only for slow repair. It is needed for 10-15% of DSBs.

46
Q

Is NHEJ and error free mechanism?

A
  • No, can be error prone, leading to chromosome translocations, deletions, mutations etc.
47
Q

SIDE NOTES

A
  • 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.