Week 5 L3: DNA REPAIR Flashcards
WHAT TYPE OF REPAIR WILL BE FOCUSED ON HERE?
Nucleotide excision repair (NER)
What is the basic principle of NER?
Nucleotides on either side of the damage are excised (one 5’ direction and one 3’ direction) and DNA synthesis repairs
Why is this the most important method?
most general
recognise bulky components
doesn’t have to have a component predisposed to that damage
recognises anything wrong
What is the main enzyme in in E.coli for NER, cleaving fragment?
UvrABC
CLEAVES THE FRAGMENT FORM dna
What are the proteins in UvrABC?
Uvr, A, B,C
What enzymes are needed to process the gap which is left?
Helicase II (UvrD) and pol I required for catalytic turnover. ligase to seal it at the end
What proteins form the functional complex of UrvABC system?
B’s ATPase activity becomes activated by A.
What protein recognises damage in DNA?
UrvA
What are some features of UrvA?
- 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
What protein of UrvABC has nuclease activity?
C
Does UrvC bind to ss DNA specifically?
NO
Does C interact with A and B in solu?
NO
What protein has ATPase activity?
B
What is the first complex to bind to DNA?
UrvAB 2A 1 or 2 B
What does complex AB do when bound to DNA?
HYDROLYSES atp AND MAKES A BIG DISTORTION IN THE DNA,
DNA WRAPS AROUND URVB
giant kink in DNA
What binds once DNA wraps around UrvB?
C (nuclease)
How and where does C cleave DNA?
dual incisions
cuts 8 nt 5’
4 nt 3’ to damage
What works on DNA after UrvC cleaves fragment?
UrvD/helicase ii
How and where does UrvD
removes the fragment
12 nt fragment
Is UrvB still present when helicase cleaves DNA?
YES
What provides the E for helicase II activity?
ATP hydrolysis
What is the role of Pol 1?
displaces UrvB and synthesise new strand. Ligase anneals the nick to existing DNA
What sis the difference in NER between bacteria and humans (eukaryotes)?
- similar just more proteins subunits involved.
- excised fragment excised is nearly 30 nt
What are the effects if NER cannot be carried out in humans?
Xeroderma pigmentosum
What are the effects of Xeroderma pigmentosum?
Hypermutability of cells on exposure to UV radiation
High frequency of light-induced skin cancer.
What did Xeroderma pigmentosum allow us to discover?
identify important genes in the human NER
the genes are called XP…
What human XP genes are involved in the recognition of the damage in the first place?
XPA, XPE
What XP gene acts like UvrB?
XZPB and D
What are the XP nucleases?
F and G
In what regions are bulky lesions repaired more rapidly?
more rapidly in transcribed regions than in silent ones
What strand has an enhanced rate of repair?
transcribed strand
What blocks repair in transcribed strand?
RNA polymerase
What is needed to overcome hinderance of RNA polymerase?
coupling factor
What is the protein that deals with coupling in DNA?
TRCF
transcription-repair coupling factor
What organisms is TRCF found in?
E.coli
What motifs does TRCF contain?
Helicase
What is the mechanism of TRCF?
- TRCF recognises stalled RNAP
- Binds to stalled complex, displacing RNAP
- Binds UvrAB in A2B complex
- Accelerates DNA repair
What are the Transcription-repair coupling genes in humans and their proteins?
ERCC8 (CSA PROETIN)
ERCC6 (CSB PROTEIN)
What XP proteins does CS protein enhance?
XPB XPD
What does the CS come from?
Mutations lead to Cockayne’s syndrome.
syndrome with defects in transcription repair coupling/
What are the symptoms of Cockayne’s syndrome?
moderate photosensitivity
serious developmental
neurological abnormalities
What does CSB protein associate with and what does this result in?
RNA pol II. this is how the repair machinery couples to transcription of mRNA in EUK.
