Lecture 7 - nucleotide excision repair

Question 1

What is nucleotide excision repair?

Answer 1

Removal of damage as a short oligonucleotide

Question 2

What are the 3 types of excision repair?

Answer 2

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

Question 3

In NER what enzyme excises the strand and what enzyme fills the resulting gap?

Answer 3

Helicase

DNA polymerase

Question 4

What are the genes responsible for conferring resistance to damage?

Answer 4

uvr (ultraviolet resistance)

Question 5

What can NER fix?

Answer 5

Cyclobutane pyrimidine dimers
(6-4) photoproduct
Chemical adducts
DNA cross links

Question 6

Describe NER in bacteria

Answer 6

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.

Question 7

Bacterial NER proteins: UvrA

Answer 7

Binds UV-irradiated dsDNA; can dimerise and complex with UvrB; has 2 ATP binding sites and 2 zinc fingers

Question 8

Bacterial NER proteins: UvrB

Answer 8

Binds UvrA2: 5’-3’ DNA helicase activity as UvrA2B2 complex.

1 ATP binding site

Question 9

Bacterial NER proteins: UvrC

Answer 9

Interacts with UvrB-DNA complex; 2 endonuclease active sites; makes 5’ & 3’ incisions

Question 10

Bacterial NER proteins: UvrD

Answer 10

3’-5’ DNA helicase
Binds ATP and DNA
releases oligonucleotide containing damaged bases

Question 11

Bacterial NER proteins: Mfd

Answer 11

Specificity for transcribed strand repair by UvrABC
displaces RNA polymerase
Interacts with UvrA

Question 12

Bacterial NER proteins: PolA

Answer 12

DNA polymerase I

Fills in the ssDNA gap left by release of the excised oligonucleotide

Question 13

Bacterial NER proteins: LigA

Answer 13

Seals the nick to complete the repair process

Question 14

Describe the steps to repairing an interstrand cross link

Answer 14

Interstrand cross link identified
NER
helicases & exonucleases expand the gap 
Homologous recombination 
NER
Fill the gap and seal the nick

Question 15

Translesion bypass DNA polymerases

Answer 15

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.

Question 16

What are the error prone DNA polymerases?

Answer 16

DNA polymerases that bypass lesions by inserting any nucleotide at the damaged site.
Polymerase IV
Polymerase V

Question 17

How many DNA polymerases does E.coli have?

Answer 17

5
3 high fidelity enzymes (I,II,III)
2 error prone enzymes (IV,V)

Question 18

GCR

Answer 18

Global genomic repair

Random search for lesions in DNA

Question 19

What genes are induced as part of the SOS response?

Answer 19

UvrABC

These genes are involved in DNA repair whose expression is stimulated following DNA damage

Question 20

During global NER what is the DNA lesion recognised by?

Answer 20

complex of UvrA and UvrB

UvrA will load UvrB onto the DNA then dissociate to leave a stable UvrB-DNA pre-incision complex

Question 21

During global NER how are UvrC and UvrD involved?

Answer 21

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)

Question 22

During global NER what fills the gap created by UvrD?

Answer 22

DNA polymerase I

THis displaces UvrB and DNA ligase completes the repair by sealing the nick at the 3’ end of the repair

Question 23

Why is UvrA a molecular matchmaker?

Answer 23

It loads UvrB onto the damaged DNA but does not take part in the remainder of the reaction

Question 24

How does UvrB prepair the DNA for UvrC?

Answer 24

UvrB bends the DNA int a beta-hairpin which is inserted between the two strands of DNA

Question 25

What is transcriptional coupled repair?

Answer 25

Describes the preferential repair of transcribed strands

Question 26

DNA lesions that impede DNA & RNA polymerases can kill cells by:

Answer 26

Interfering with DNA replication in actively growing and dividing cells
Blocking transcription and depriving cells of an essential protein

Question 27

Describe transcription coupled repair

Answer 27

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)

Question 28

Mutation frequency decline

Answer 28

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.

Question 29

What happens when RNA polymerase is stalled at a lesion?

Answer 29

It is difficult for the damage to be identified by NER factors (UvrA & UvrB) so prevents repair.

Question 30

What recognises RNA polymerase stalled at a lesion?

Answer 30

Mfd- this binds a beta subunit of RNA polymerase and ejects RNA polymerase and the transcript from the DNA. UvrA is recruited…