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Flashcards in DSB 2 Deck (47)
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1

What are the 2 major DSB repair pathways?

1. NHEJ
2. HR

2

What is non homologous end joining?

- Ligating broken ends of DNA
- Doesn't require homology
- Error prone process

3

At what stage in the cell cycle does NHEJ function?

G0 and G1 phase of the cell cycle

4

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

5

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

6

What is the KU complex?

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

7

What does the KU complex do?

Protects DNA ends from nucleolytic degradation and other aberrant processing

8

What is DNA PKcs?

- It is a PIKK
- Very large protein- 469 KDa

9

Name 2 other members of the PIKK family

ATM and ATR

10

What is CDNA?

DNA-PKcs

11

How big is CDNA?

14 KB

12

Describe the structure of DNA PKcs

- Has lots of alpha helical HEAT motifs to allow bending and folding into hollow crystal structure

13

What is the most prevalent DSB repair?

Phosphorylation by histone H2AX

14

What is the role of histones?

To allow DNA to be compact in cells

15

What makes up a nucleosome?

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

16

How many base pairs does each histone octamer coat?

150

17

Name a variant of H2A

H2AX

18

What are histones phosphorylated on?

N terminal tail

19

Name a substrate of DNA-PKcs

- H2A

20

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

21

How is the release of DNA PKcs from KU regulated?

DNA PK has 3 clusters of auto-phosphorylation sites

22

What is artemis?

A nuclease involved in NHEJ using VDJ recombination

23

What does DNA PK activate?

artemis, XRCC4 and DNA ligase 4

24

What is processing actually for?

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