Week 5 L3: DNA REPAIR

Question 1

WHAT TYPE OF REPAIR WILL BE FOCUSED ON HERE?

Answer 1

Nucleotide excision repair (NER)

Question 2

What is the basic principle of NER?

Answer 2

Nucleotides on either side of the damage are excised (one 5’ direction and one 3’ direction) and DNA synthesis repairs

Question 3

Why is this the most important method?

Answer 3

most general
recognise bulky components
doesn’t have to have a component predisposed to that damage
recognises anything wrong

Question 4

What is the main enzyme in in E.coli for NER, cleaving fragment?

Answer 4

UvrABC

CLEAVES THE FRAGMENT FORM dna

Question 5

What are the proteins in UvrABC?

Answer 5

Uvr, A, B,C

Question 6

What enzymes are needed to process the gap which is left?

Answer 6

Helicase II (UvrD) and pol I required for catalytic turnover. ligase to seal it at the end

Question 7

What proteins form the functional complex of UrvABC system?

Answer 7

B’s ATPase activity becomes activated by A.

Question 8

What protein recognises damage in DNA?

Answer 8

UrvA

Question 9

What are some features of UrvA?

Answer 9

• 103 kDa ATPase that recognises damage
• 2 Zn fingers
• 2 ATP binding sites
• binds damaged DNA Ks = 107 - 1011 M-
• eg TT cyclobutane 2 x better than normal DNA, but
  6-4 photoproduct 1000 x tighter

Question 10

What protein of UrvABC has nuclease activity?

Answer 10

C

Question 11

Does UrvC bind to ss DNA specifically?

Answer 11

NO

Question 12

Does C interact with A and B in solu?

Answer 12

NO

Question 13

What protein has ATPase activity?

Answer 13

B

Question 14

What is the first complex to bind to DNA?

Answer 14

UrvAB 2A 1 or 2 B

Question 15

What does complex AB do when bound to DNA?

Answer 15

HYDROLYSES atp AND MAKES A BIG DISTORTION IN THE DNA,
DNA WRAPS AROUND URVB
giant kink in DNA

Question 16

What binds once DNA wraps around UrvB?

Answer 16

C (nuclease)

Question 17

How and where does C cleave DNA?

Answer 17

dual incisions
cuts 8 nt 5’
4 nt 3’ to damage

Question 18

What works on DNA after UrvC cleaves fragment?

Answer 18

UrvD/helicase ii

Question 19

How and where does UrvD

Answer 19

removes the fragment

12 nt fragment

Question 20

Is UrvB still present when helicase cleaves DNA?

Answer 20

YES

Question 21

What provides the E for helicase II activity?

Answer 21

ATP hydrolysis

Question 22

What is the role of Pol 1?

Answer 22

displaces UrvB and synthesise new strand. Ligase anneals the nick to existing DNA

Question 23

What sis the difference in NER between bacteria and humans (eukaryotes)?

Answer 23

• similar just more proteins subunits involved.

- excised fragment excised is nearly 30 nt

Question 24

What are the effects if NER cannot be carried out in humans?

Answer 24

Xeroderma pigmentosum

Question 25

What are the effects of Xeroderma pigmentosum?

Answer 25

Hypermutability of cells on exposure to UV radiation | High frequency of light-induced skin cancer.

Question 26

What did Xeroderma pigmentosum allow us to discover?

Answer 26

identify important genes in the human NER | the genes are called XP...

Question 27

What human XP genes are involved in the recognition of the damage in the first place?

Answer 27

XPA, XPE

Question 28

What XP gene acts like UvrB?

Answer 28

XZPB and D

Question 29

What are the XP nucleases?

Answer 29

F and G

Question 30

In what regions are bulky lesions repaired more rapidly?

Answer 30

more rapidly in transcribed regions than in silent ones

Question 31

What strand has an enhanced rate of repair?

Answer 31

transcribed strand

Question 32

What blocks repair in transcribed strand?

Answer 32

RNA polymerase

Question 33

What is needed to overcome hinderance of RNA polymerase?

Answer 33

coupling factor

Question 34

What is the protein that deals with coupling in DNA?

Answer 34

TRCF | transcription-repair coupling factor

Question 35

What organisms is TRCF found in?

Answer 35

E.coli

Question 36

What motifs does TRCF contain?

Answer 36

Helicase

Question 37

What is the mechanism of TRCF?

Answer 37

- TRCF recognises stalled RNAP - Binds to stalled complex, displacing RNAP - Binds UvrAB in A2B complex - Accelerates DNA repair

Question 38

What are the Transcription-repair coupling genes in humans and their proteins?

Answer 38

ERCC8 (CSA PROETIN) | ERCC6 (CSB PROTEIN)

Question 39

What XP proteins does CS protein enhance?

Answer 39

XPB XPD

Question 40

What does the CS come from?

Answer 40

Mutations lead to Cockayne’s syndrome. | syndrome with defects in transcription repair coupling/

Question 41

What are the symptoms of Cockayne's syndrome?

Answer 41

moderate photosensitivity serious developmental neurological abnormalities

Question 42

What does CSB protein associate with and what does this result in?

Answer 42

RNA pol II. this is how the repair machinery couples to transcription of mRNA in EUK.