Week 5 L2: DNA REPAIR Flashcards
What are the 3 strategies for DNA repair?
base excision, direct reversal and nucleotide excision.
What is Direct reversal of DNA damage?
e.g methylation event, simply demethylation event.
No change to backbone or synthesis
What is an example of Direct reversal?
DNA photolyase and DNA methyltransferase
What is the effect of DNA photolyase?
repairs pyrimidine dimers (cyclobutane dimer)
undoes the dimer formation
Like in thymine dimers.
crosslinking
What energy is needed for Photolyase to repair pyrimidine dimers?
Photoreactivating
uses light in a photochemical reaction
Is the Photolyase reaction fast?
yes
What is the structure of photolyase?
two tightly-bound chromophores (one is always FASH2)
What is the mechanism of photolyase?
- 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
What does the enzyme photolyase bind to?
DNA containing pyrimidine dimer
What wavelength does photolyase absorb?
visible light 300-500nm, using second chromophore
What is energy transferred to in photolyase?
FADH2
What is the role of FADH2 in photolyase?
transfers e- to pyrimidine dimer
What is the fundamental step in the splitting of pyrimidine dimer?
the e- transfer causes the dimer to split into a monomer
What is the chromophore?
part of the molecule responsible for colour
What are the major substrates of DNA-methyltransferase?
O6 methylguanine
O4 methylthymine
What is the role of DNA methyltransferase?
repair methyl damage to DNA
How does DNA-methyltransferase demethylate? e.g. ada protein
removes the alkyl groups from oxygen bases
Transfer methyl to a nucleophilic cystine in the protein C321 residue
(METHYL TRANSFER)
What is an example of a methyltransferase in E.coli?
Ada protein
from ada gene
What is the structure of the ada protein?
CONSTRUCTED IN 2 HALVES
What terminus of the ada protein repairs O6 methylguanine?
C terminus
How does the methyltransferase remove the methyl (what motion)?
sucking it into the enzyme
flip the base out of the helix into a pocket in the protein where chemistry can occur
BASE FLIPPING
What is an example of oxidative demethylation enzyme?
ENZYME: dioxygenases
iron dependent reaction with molecular oxygen.
What is the reaction mechanism of oxidative demethylation?
iron dependent reaction with molecular oxygen.
1-METHYLADENANINE Oxygen removes methyl from nitrogen at 1 position and converts to formaldehide. with bioproduct of succinate
What is the next DNA base repair method we will talk about?
BASE EXCISION
What is base excision?
removing just that nucleotide from the DNA and replacing it with a new one by repair synthesis
What base excision example will we look at?
deoxyuracil
uracil base instead of thymine
What group does deoxyuracil lack that thymine does have?
5’ methyl group
What enzyme removes the uracil base from deoxyuracil?
uracil glycosylase
What does uracil glycosylase produce?
reacts only with nuclear base (uracil)
a-basic site, only a sugar with no base
What does AP endonuclease do to the sugar and phosphate left?
breaks the phosphodiester linkage on the 5’ side of the 5’ phosphate.
break in backbone at position 3
What are the 2 nucleases in base excision repair?
AP endonuclease and 5’-phosphodiesterase
What does 5’phosphodiesterase do?
It reacts on other side of a-basic sugar
complete removal of nucleotide. (bottom link)
What does DNA polymerase introduce into the base excision mechanism?
TTP (thymine) and 5’ phosphate linked
What is the final step/ enzyme in the base excision pathway?
DNA ligase to connect the 3’ hydroxyl to the next 5’ phosphate.
base has thymine instead of uracil and repaid is compete
What is the key step (give specificity in the base excision pathway?
first step involving glycosylase
Is glycosylase specific?
YES
each type of damaged nucleobase requires its own glycosylase
e.g uracil as shown, thymine glycol… and more
What DNA glycosylase will we look at?
Uracil glycosylase
present in human and bacteria
What is the basic role of uracil glycosylase?
Switch uracil to thymine as DNA does not contain uracil
What does Uracil glycosylase cleave in DNA?
cleaves N-glycosyl bond only
Does DNA uracil glycosylase work on RNA?
NO
NO activity in RNA
How does Uracil and glycosylase interact?
Uracil binding site in narrow channel at centre of protein (active centre)
H bonding interactions with uracil.
How does glycosylase interact with uracil (more specific) (4 interactions)?
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.
How is uracil glycosylase specific to DNA?
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
does base flipping occur in glycosylase?
YES the structure suggests it is probable
What discriminates 8-oxoguanine in DNA from normal guanine? How does glycosylase differ between the 2?
Gly-42
How does 8-oxoG and guanine differ? how does glycosylase differ between?
proton on N7 of 8-oxo, is accepted by gly42
How does Lys249 kick out 8-oxoG?
attacks the glycosyl carbon and forms a bond.
leaving abasic site
What is the structure of AP-endonuclease?
cleave to abasic site
Crystal structure of Exonuclease III
4-layered alpha-beta-sandwich
Proposed nucleophile is
Asp - His - H2O triad
