L5 - Excision repair

Question 1

what is excision repair

Answer 1

cutting out DNA and replacing it

Question 2

what is the general strategy of excision repair

Answer 2

1. find damage
2. cut on both sides of damage
3. remove damaged DNA
4. copy undamged strand to make patch

Question 3

difference in removal of large vs small sequnce removal of damaged DNA in exision pathways

Answer 3

small sequnces will simply drift away due to low strength of H bond

large sequences require motor proteins

Question 4

general mechansism for base excision repair

Answer 4

1. DNA glycosylases scan DNA and find damage
2. cleave N-glycosyl bond = abasic site
3. AP endonuclease cleaves backbone on 5’ side of damage = leave sgap with open 3’ hydroxyl
4. DNA polymerase adds nucleotide
5. DRP lyase (deoxyribose phosphate lyase) cleaves backbone on 3’ side of flap

= flap released
6. DNA ligase heals nick

Question 5

name of enzyme involved in repair of base-excision repair

Answer 5

DNA-glycosylases

= different ones for each form of damage
= all produce abasic sites so after cleavage all pathways are the same

Question 6

how are damaged bases recognised in huge genome

Answer 6

base flipping

damaged DNA is more unstable than undamaged
= flips 180˚ into active site of glycosylases
= no ATP required just more energetically stable

Question 7

describve the genral mechanism of Nucelotide excision repair in bacteria

Answer 7

1. bulky damage recognised by UvrA + UvrB complex
2. UvrA distorts/bends the DNA
3. UvrB moves along DNA until it cant move = bulky lesion = comfirmed damage
4. UvrA disociates after comfirmation allowing UvrC to be recruited to UvrB
5. UvrC cleaves base 4-5 bases away on 3’ side of damage
6. cleave 8 bases 5’ of damage
7. UvrD helicase removes the 12-13 oglionucleotide WITH the UvrC
   8 Polymerase and ligase fill gap

Question 8

compare the BER and NER pathways

Answer 8

BER:
- non-bulky
- intrinsic damage
- repair patch is 1 nucleotide long
- specifc enzyme per type of damage

NER:
- bulky esions
- extrinsic damage
- large repair patches
- broad specificity = enzymes recognise differnt lesions

Question 9

name 1 accidental and 1 programmed reason for double strand breaks

Answer 9

accidental = ionising radiation
- chromosone instability
= can be lethal

programmed = V(D)J recombination
- beneficial genetic variation = antibodies

Question 10

double vs single end dsDNA breaks

Answer 10

double end:
clean break with 2 blunt ends

single end:
replication over nick in single strand can cause

Question 11

descrive the mechanism of non-homologous end joining

Answer 11

Only 1 copy of DNA required to fix = 2 in homologous recombination

1. The 2 ends are tethered together by ku 70/80 complex = end binding
2. recruits DNA-PKcs
3. recruits artemis with endonucleases that proscess the ends = 3’ overhangs
4. complex-containing-ligase binds and continously adds and removes nucleotides

= repaired but wioth altered DNA sequnce

Question 12

what point in the cell cycle can Non-homologous end-joining (NHEJ) occur

Answer 12

any stage in cell cycle

= Homologous recombination only in S phase = when there are 2 copis of same DNA present

Question 13

summary of NHEJ

Answer 13

no requirement for homologous donor DNA

no stage in cell cyle

introduces random nucleotides at break site from end proscessing

= common in eukayotes but rare in bacteria