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Flashcards in NER 2 Deck (35)
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1

What are XP proteins involved in?

- Damage recognition
- Unwinding of DNA
- Cutting either side of the damage

2

What are the subunits of TFIIH?

- XPD
- XPB
- TTDA
(TFIIH encoded by the genes of the proteins above)

3

What is the role of TFIIH?

Has roles in both nucleotide excision repair and transcription

4

What is TTD?

Trichothiodystrophy is a transcription syndrome

5

What do CS proteins recruit and why?

- NER proteins = enable NER at the site where RNA polymerase is stalled at the damage

- Chromatin remodellers = enable RNA polymerase to be pushed back so that NER proteins can be recruited to the site of damage on the mRNA

6

What are the first steps of GG-NER pathway (up to damage verification)? STEP 1

1. XPE-DDB1 then XPC recognises the damage

2. TFIIH recruited to the site of damage, which opens up the structure

3. XPA-RPA helicase recruited to the site of damage to verify the damage and to position everything properly

7

What happens after damage verification in GG-NER pathway? STEP 2

4. XPG nuclease cuts the damage at the 3' site independently of XPA but dependant on XPC

5. ERCC1 and XPF heterodimer nuclease cuts the damage at 5' site

8

What are the last steps of the GG-NER pathway (after cutting at sites)? STEP 3

6. Cleavage of the backbone of DNA, removing 25-30 bases

7. DNA polymerase fills the gap

8. DNA ligase seals everything

9

What is XPC and what does it do?

It is a general recognition factor and it must detect 1 damaged base in 1 million undamaged bases

10

What is not well recognized by XPC?

CPD (64PP is recognized well?)

11

What is DDB?

It is a heterodimer made up of DDB1 and DDB2, this makes up the whole XPE protein

12

What is the structure of DDB1?

DDB1 = 3 domains

13

What does DDB2 do?

It is part of the XP protein that recognised the damage in DNA

14

What does DDB actually do?

- Binds to damage, rotates and ubiquitinates chromatin proteins

- Ubiquitinates and recruits XPC to damaged site

- DDB also self ubiquitinates but this leads to its own degredation

15

Is XPE expressed in rodent cells?

NO.

16

Are human mutated XPE cells sensitive to UV?

They are only midly sensitive to killing by UV

17

What is the structure of XPC/RAD4?

- Has lots of domains
- One part binds to the double stranded DNA?
- BHD (beta hairpin domain) binds to the unpaired bases on the other side?????

18

What is TFIIH?

It is a transcription initiation factor

19

How many subunits does TFIIH have?

10

20

Name a subunit of TFIIH?

XPB

21

How was XPB discovered?

1. Isolated P89
2. Gene was cloned
3. Then it was sequenced
= turned out to be XPB

22

What does TFIIH actually do?

- Opens up promotor site in transcription
- Has helicase activity but this isn't actually needed in NER

23

What is XPA homologous to?

Yeast RAD14

24

What do XPA and XPC knockout mice show?

Sensitivity to UV light and susceptibility to UV carcinogenesis

25

What does XPA bind strongly to?

To UV irradiated DNA

26

What actually is XPA?

It is a zinc finger protein

27

What proteins in the NER pathway are vital for survival in mice?

ERCC1, XPF, XPG
Because when KO in mice, they barely survive

28

What does ERCC1 need to work?

XPA and XPC

29

Describe the first mechanism to repair damage in the TC-NER pathway in E.coli

1. RNA polymerase gets to site of damage and gets blocked (can't move futher and stops NER proteins from fixing damage)

2. Recruitment of TRCF (transcription repair coupling protein) by RNA pol

3. TRCF dissociates from RNA pol and mRNA comes off DNA = empty DNA strand

4. TRCF recruits UVR A2B2 heterotetrimer complex

5. UVRB recruited damage on mRNA, then recruits UVRC to do its thing then whole NER pathway will happen

30

Describe the alternative mechansim of TC-NER pathway

1. UVRD helicase and NusA push back the RNA polymerase
2. UVRA and UVRB come and reapir the damge