Lecture 7 - nucleotide excision repair Flashcards
What is nucleotide excision repair?
Removal of damage as a short oligonucleotide
What are the 3 types of excision repair?
Mis-match repair: removal of the newly synthesised strand (that contains the mis-match) after replication
Base excision repair: removal of damaged bases
Nucleotide excision repair: removal of damage as a short oligonucleotide
In NER what enzyme excises the strand and what enzyme fills the resulting gap?
Helicase
DNA polymerase
What are the genes responsible for conferring resistance to damage?
uvr (ultraviolet resistance)
What can NER fix?
Cyclobutane pyrimidine dimers
(6-4) photoproduct
Chemical adducts
DNA cross links
Describe NER in bacteria
Incisions are made 12-13 nucleotides apart
A 12- nucleotide strand is removed with a monoadduct and a 13-nucleotide strand with a di-adduct. The 5’ cut site is fixed at the 8th phosphodiester bond 5’ of the damaged site. The 3’ cut site is at the 4th or 5th bond 3’ of the damaged site.
The gap is filled by DNA polymerase I.
Bacterial NER proteins: UvrA
Binds UV-irradiated dsDNA; can dimerise and complex with UvrB; has 2 ATP binding sites and 2 zinc fingers
Bacterial NER proteins: UvrB
Binds UvrA2: 5’-3’ DNA helicase activity as UvrA2B2 complex.
1 ATP binding site
Bacterial NER proteins: UvrC
Interacts with UvrB-DNA complex; 2 endonuclease active sites; makes 5’ & 3’ incisions
Bacterial NER proteins: UvrD
3’-5’ DNA helicase
Binds ATP and DNA
releases oligonucleotide containing damaged bases
Bacterial NER proteins: Mfd
Specificity for transcribed strand repair by UvrABC
displaces RNA polymerase
Interacts with UvrA
Bacterial NER proteins: PolA
DNA polymerase I
Fills in the ssDNA gap left by release of the excised oligonucleotide
Bacterial NER proteins: LigA
Seals the nick to complete the repair process
Describe the steps to repairing an interstrand cross link
Interstrand cross link identified NER helicases & exonucleases expand the gap Homologous recombination NER Fill the gap and seal the nick
Translesion bypass DNA polymerases
E.coli has two DNA polymerases that can bypass lesions by inserting any nucleotide at the damaged site. The polymerases are: Polymerase IV (DinB) PolV (UmuD'2C) This type of DNA synthesis only occurs when cells are so damaged that managing to replicate and transcribe is more important than the risk of generating lots of mutations.
What are the error prone DNA polymerases?
DNA polymerases that bypass lesions by inserting any nucleotide at the damaged site.
Polymerase IV
Polymerase V
How many DNA polymerases does E.coli have?
5
3 high fidelity enzymes (I,II,III)
2 error prone enzymes (IV,V)
GCR
Global genomic repair
Random search for lesions in DNA
What genes are induced as part of the SOS response?
UvrABC
These genes are involved in DNA repair whose expression is stimulated following DNA damage
During global NER what is the DNA lesion recognised by?
complex of UvrA and UvrB
UvrA will load UvrB onto the DNA then dissociate to leave a stable UvrB-DNA pre-incision complex
During global NER how are UvrC and UvrD involved?
UvrC is recruited by UvrB and cleaves the damaged strand on both sides of the lesion. UvrD (DNA helicase III) displaces UvrC and the 12-13 nucleotide section containing the damages bas(s)
During global NER what fills the gap created by UvrD?
DNA polymerase I
THis displaces UvrB and DNA ligase completes the repair by sealing the nick at the 3’ end of the repair
Why is UvrA a molecular matchmaker?
It loads UvrB onto the damaged DNA but does not take part in the remainder of the reaction
How does UvrB prepair the DNA for UvrC?
UvrB bends the DNA int a beta-hairpin which is inserted between the two strands of DNA
What is transcriptional coupled repair?
Describes the preferential repair of transcribed strands
DNA lesions that impede DNA & RNA polymerases can kill cells by:
Interfering with DNA replication in actively growing and dividing cells
Blocking transcription and depriving cells of an essential protein
Describe transcription coupled repair
A system that targets DNA repair enzymes to transcriotionally active gene
In bacteria RNA polymerase stalled at a lesion is recognised by an enzyme called Mfd (mutation frequency decline) also known as transcription coupling factor (TRCF)
Mutation frequency decline
Holding bacteria cells in poor medium inhibited protein synthesis temporarily gave the cells time to repair damage from UV light and preferentially on the transcribed strand.
Cells defective in Mfd gene do not exhibit mutation, frequency decline and a link to a NER was uncovered.
What happens when RNA polymerase is stalled at a lesion?
It is difficult for the damage to be identified by NER factors (UvrA & UvrB) so prevents repair.
What recognises RNA polymerase stalled at a lesion?
Mfd- this binds a beta subunit of RNA polymerase and ejects RNA polymerase and the transcript from the DNA. UvrA is recruited…
