NER 2

Question 1

What are XP proteins involved in?

Answer 1

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

Question 2

What are the subunits of TFIIH?

Answer 2

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

Question 3

What is the role of TFIIH?

Answer 3

Has roles in both nucleotide excision repair and transcription

Question 4

What is TTD?

Answer 4

Trichothiodystrophy is a transcription syndrome

Question 5

What do CS proteins recruit and why?

Answer 5

• 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

Question 6

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

Answer 6

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

Question 7

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

Answer 7

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

Question 8

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

Answer 8

6. Cleavage of the backbone of DNA, removing 25-30 bases
7. DNA polymerase fills the gap
8. DNA ligase seals everything

Question 9

What is XPC and what does it do?

Answer 9

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

Question 10

What is not well recognized by XPC?

Answer 10

CPD (64PP is recognized well?)

Question 11

What is DDB?

Answer 11

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

Question 12

What is the structure of DDB1?

Answer 12

DDB1 = 3 domains

Question 13

What does DDB2 do?

Answer 13

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

Question 14

What does DDB actually do?

Answer 14

• 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

Question 15

Is XPE expressed in rodent cells?

Answer 15

NO.

Question 16

Are human mutated XPE cells sensitive to UV?

Answer 16

They are only midly sensitive to killing by UV

Question 17

What is the structure of XPC/RAD4?

Answer 17

• 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?????

Question 18

What is TFIIH?

Answer 18

It is a transcription initiation factor

Question 19

How many subunits does TFIIH have?

Answer 19

10

Question 20

Name a subunit of TFIIH?

Answer 20

XPB

Question 21

How was XPB discovered?

Answer 21

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

Question 22

What does TFIIH actually do?

Answer 22

• Opens up promotor site in transcription

- Has helicase activity but this isn’t actually needed in NER

Question 23

What is XPA homologous to?

Answer 23

Yeast RAD14

Question 24

What do XPA and XPC knockout mice show?

Answer 24

Sensitivity to UV light and susceptibility to UV carcinogenesis

Question 25

What does XPA bind strongly to?

Answer 25

To UV irradiated DNA

Question 26

What actually is XPA?

Answer 26

It is a zinc finger protein

Question 27

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

Answer 27

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

Question 28

What does ERCC1 need to work?

Answer 28

XPA and XPC

Question 29

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

Answer 29

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

Question 30

Describe the alternative mechansim of TC-NER pathway

Answer 30

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

Question 31

What is XPD?

Answer 31

A helicase

Question 32

How many domains does XPD have?

Answer 32

7 conserved domains

Question 33

What do XPD mutations cause?

Answer 33

- XP = affects the activity of the protein which would affect transcription - TTD = affects the stability of the complex itself

Question 34

Describe TTD symptoms in mice?

Answer 34

Stunted growth and hair loss due to missing base in XPD gene

Question 35

Describe the TC-NER pathway in humans

Answer 35

1. RNApol II reaches site of damage 2. Weakly bound to CSB and XPG protein 3. RNApol gets blocked by damage = this is a signal for TC-NER 4. CSB proteins tightly bind to RNApol = recruits chromatin remodellers (around histones/non-histone proteins) that recruit CSA 5. Remodellers removes proteins behind RNApol = can be pushed back = Now NER proteins can be recruited to carry on rest of NER process independantly of XPC/XPE