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L3 Gene Regulation > Week 5 L2: DNA REPAIR > Flashcards

Week 5 L2: DNA REPAIR Flashcards

1
Q

What are the 3 strategies for DNA repair?

A

base excision, direct reversal and nucleotide excision.

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2
Q

What is Direct reversal of DNA damage?

A

e.g methylation event, simply demethylation event.

No change to backbone or synthesis

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3
Q

What is an example of Direct reversal?

A

DNA photolyase and DNA methyltransferase

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4
Q

What is the effect of DNA photolyase?

A

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

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5
Q

What energy is needed for Photolyase to repair pyrimidine dimers?

A

Photoreactivating

uses light in a photochemical reaction

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6
Q

Is the Photolyase reaction fast?

A

yes

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7
Q

What is the structure of photolyase?

A

two tightly-bound chromophores (one is always FASH2)

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8
Q

What is the mechanism of photolyase?

A
  • 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
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9
Q

What does the enzyme photolyase bind to?

A

DNA containing pyrimidine dimer

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10
Q

What wavelength does photolyase absorb?

A

visible light 300-500nm, using second chromophore

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11
Q

What is energy transferred to in photolyase?

A

FADH2

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12
Q

What is the role of FADH2 in photolyase?

A

transfers e- to pyrimidine dimer

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13
Q

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

A

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

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14
Q

What is the chromophore?

A

part of the molecule responsible for colour

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15
Q

What are the major substrates of DNA-methyltransferase?

A

O6 methylguanine

O4 methylthymine

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16
Q
A
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17
Q

What is the role of DNA methyltransferase?

A

repair methyl damage to DNA

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18
Q

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

A

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

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19
Q

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

A

Ada protein

from ada gene

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20
Q

What is the structure of the ada protein?

A

CONSTRUCTED IN 2 HALVES

21
Q

What terminus of the ada protein repairs O6 methylguanine?

A

C terminus

22
Q

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

A

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

23
Q

What is an example of oxidative demethylation enzyme?

A

ENZYME: dioxygenases

iron dependent reaction with molecular oxygen.

24
Q

What is the reaction mechanism of oxidative demethylation?

A

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

25
Q

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

A

BASE EXCISION

26
Q

What is base excision?

A

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

27
Q

What base excision example will we look at?

A

deoxyuracil

uracil base instead of thymine

28
Q

What group does deoxyuracil lack that thymine does have?

A

5’ methyl group

29
Q

What enzyme removes the uracil base from deoxyuracil?

A

uracil glycosylase

30
Q

What does uracil glycosylase produce?

A

reacts only with nuclear base (uracil)

a-basic site, only a sugar with no base

31
Q

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

A

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

32
Q

What are the 2 nucleases in base excision repair?

A

AP endonuclease and 5’-phosphodiesterase

33
Q

What does 5’phosphodiesterase do?

A

It reacts on other side of a-basic sugar

complete removal of nucleotide. (bottom link)

34
Q

What does DNA polymerase introduce into the base excision mechanism?

A

TTP (thymine) and 5’ phosphate linked

35
Q

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

A

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

36
Q

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

A

first step involving glycosylase

37
Q

Is glycosylase specific?

A

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

38
Q

What DNA glycosylase will we look at?

A

Uracil glycosylase

present in human and bacteria

39
Q

What is the basic role of uracil glycosylase?

A

Switch uracil to thymine as DNA does not contain uracil

40
Q

What does Uracil glycosylase cleave in DNA?

A

cleaves N-glycosyl bond only

41
Q

Does DNA uracil glycosylase work on RNA?

A

NO

NO activity in RNA

42
Q

How does Uracil and glycosylase interact?

A

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

43
Q

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

A

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.

44
Q

How is uracil glycosylase specific to DNA?

A 
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
45
Q

does base flipping occur in glycosylase?

A

YES the structure suggests it is probable

46
Q

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

A

Gly-42

47
Q

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

A

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

48
Q

How does Lys249 kick out 8-oxoG?

A

attacks the glycosyl carbon and forms a bond.

leaving abasic site

49
Q

What is the structure of AP-endonuclease?

A

cleave to abasic site
Crystal structure of Exonuclease III

4-layered alpha-beta-sandwich

Proposed nucleophile is
Asp - His - H2O triad

L3 Gene Regulation (14 decks)

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