Week 5 L2: DNA REPAIR

Question 1

What are the 3 strategies for DNA repair?

Answer 1

base excision, direct reversal and nucleotide excision.

Question 2

What is Direct reversal of DNA damage?

Answer 2

e.g methylation event, simply demethylation event.

No change to backbone or synthesis

Question 3

What is an example of Direct reversal?

Answer 3

DNA photolyase and DNA methyltransferase

Question 4

What is the effect of DNA photolyase?

Answer 4

repairs pyrimidine dimers (cyclobutane dimer)
undoes the dimer formation
Like in thymine dimers.
crosslinking

Question 5

What energy is needed for Photolyase to repair pyrimidine dimers?

Answer 5

Photoreactivating

uses light in a photochemical reaction

Question 6

Is the Photolyase reaction fast?

Answer 6

yes

Question 7

What is the structure of photolyase?

Answer 7

two tightly-bound chromophores (one is always FASH2)

Question 8

What is the mechanism of photolyase?

Answer 8

• Enzyme binds to DNA containing pyrimidine dimer
• Absorbs visible light (300-500 nm), using second chromophore
• Energy transferred to FADH2
• FADH2 transfers electron to pyrimidine dimer
• pyrimidine dimer splits into monomeric form

Question 9

What does the enzyme photolyase bind to?

Answer 9

DNA containing pyrimidine dimer

Question 10

What wavelength does photolyase absorb?

Answer 10

visible light 300-500nm, using second chromophore

Question 11

What is energy transferred to in photolyase?

Answer 11

FADH2

Question 12

What is the role of FADH2 in photolyase?

Answer 12

transfers e- to pyrimidine dimer

Question 13

What is the fundamental step in the splitting of pyrimidine dimer?

Answer 13

the e- transfer causes the dimer to split into a monomer

Question 14

What is the chromophore?

Answer 14

part of the molecule responsible for colour

Question 15

What are the major substrates of DNA-methyltransferase?

Answer 15

O6 methylguanine

O4 methylthymine

Question 17

What is the role of DNA methyltransferase?

Answer 17

repair methyl damage to DNA

Question 18

How does DNA-methyltransferase demethylate? e.g. ada protein

Answer 18

removes the alkyl groups from oxygen bases
Transfer methyl to a nucleophilic cystine in the protein C321 residue
(METHYL TRANSFER)

Question 19

What is an example of a methyltransferase in E.coli?

Answer 19

Ada protein

from ada gene

Question 20

What is the structure of the ada protein?

Answer 20

CONSTRUCTED IN 2 HALVES

Question 21

What terminus of the ada protein repairs O6 methylguanine?

Answer 21

C terminus

Question 22

How does the methyltransferase remove the methyl (what motion)?

Answer 22

sucking it into the enzyme
flip the base out of the helix into a pocket in the protein where chemistry can occur
BASE FLIPPING

Question 23

What is an example of oxidative demethylation enzyme?

Answer 23

ENZYME: dioxygenases

iron dependent reaction with molecular oxygen.

Question 24

What is the reaction mechanism of oxidative demethylation?

Answer 24

iron dependent reaction with molecular oxygen.
1-METHYLADENANINE Oxygen removes methyl from nitrogen at 1 position and converts to formaldehide. with bioproduct of succinate

Question 25

What is the next DNA base repair method we will talk about?

Answer 25

BASE EXCISION

Question 26

What is base excision?

Answer 26

removing just that nucleotide from the DNA and replacing it with a new one by repair synthesis

Question 27

What base excision example will we look at?

Answer 27

deoxyuracil | uracil base instead of thymine

Question 28

What group does deoxyuracil lack that thymine does have?

Answer 28

5' methyl group

Question 29

What enzyme removes the uracil base from deoxyuracil?

Answer 29

uracil glycosylase

Question 30

What does uracil glycosylase produce?

Answer 30

reacts only with nuclear base (uracil) | a-basic site, only a sugar with no base

Question 31

What does AP endonuclease do to the sugar and phosphate left?

Answer 31

breaks the phosphodiester linkage on the 5' side of the 5' phosphate. break in backbone at position 3

Question 32

What are the 2 nucleases in base excision repair?

Answer 32

AP endonuclease and 5'-phosphodiesterase

Question 33

What does 5'phosphodiesterase do?

Answer 33

It reacts on other side of a-basic sugar | complete removal of nucleotide. (bottom link)

Question 34

What does DNA polymerase introduce into the base excision mechanism?

Answer 34

TTP (thymine) and 5' phosphate linked

Question 35

What is the final step/ enzyme in the base excision pathway?

Answer 35

DNA ligase to connect the 3' hydroxyl to the next 5' phosphate. base has thymine instead of uracil and repaid is compete

Question 36

What is the key step (give specificity in the base excision pathway?

Answer 36

first step involving glycosylase

Question 37

Is glycosylase specific?

Answer 37

YES each type of damaged nucleobase requires its own glycosylase e.g uracil as shown, thymine glycol... and more

Question 38

What DNA glycosylase will we look at?

Answer 38

Uracil glycosylase | present in human and bacteria

Question 39

What is the basic role of uracil glycosylase?

Answer 39

Switch uracil to thymine as DNA does not contain uracil

Question 40

What does Uracil glycosylase cleave in DNA?

Answer 40

cleaves N-glycosyl bond only

Question 41

Does DNA uracil glycosylase work on RNA?

Answer 41

NO | NO activity in RNA

Question 42

How does Uracil and glycosylase interact?

Answer 42

Uracil binding site in narrow channel at centre of protein (active centre) H bonding interactions with uracil.

Question 43

How does glycosylase interact with uracil (more specific) (4 interactions)?

Answer 43

H-bonds formed at both exocyclic oxygens of the uracil to 2 side chains of the glycosylase. (Q87 AND N147). Interact with 2 aromatic compounds F101 and Y90.

Question 44

How is uracil glycosylase specific to DNA?

Answer 44

``` Tyr 90 (F101 RING)sidechain excludes 5-methyl excludes thymine. Phe 101 (on the Y90)clash with 2'OH, excludes RNA Specific to Uracil in DNA ```

Question 45

does base flipping occur in glycosylase?

Answer 45

YES the structure suggests it is probable

Question 46

What discriminates 8-oxoguanine in DNA from normal guanine? How does glycosylase differ between the 2?

Answer 46

Gly-42

Question 47

How does 8-oxoG and guanine differ? how does glycosylase differ between?

Answer 47

proton on N7 of 8-oxo, is accepted by gly42

Question 48

How does Lys249 kick out 8-oxoG?

Answer 48

attacks the glycosyl carbon and forms a bond. | leaving abasic site

Question 49

What is the structure of AP-endonuclease?

Answer 49

cleave to abasic site Crystal structure of Exonuclease III 4-layered alpha-beta-sandwich Proposed nucleophile is Asp - His - H2O triad