DNA Damage & Repair

Question 1

What is a mutation?

Answer 1

When DNA damage makes it into the next generation (has to be heritable)

Question 2

How is uracil removed from DNA?

Answer 2

Base excision repair (BER)

Question 3

Dam methylase

Answer 3

Methylates specific sequence, results in E. coli being able to discern between parent and daughter strand in mismatch repair

Question 4

What is transcription coupled repair?

Answer 4

A form of NER

Question 5

What is translesion DNA synthesis?

Answer 5

DNA synthesis “past” a lesion in the sequence; frequently results in mismatches. Also called SOS repair. Involves Pol IV and V

Question 6

What are some harmful physical agents?

Answer 6

Heat, X-ray, UV

Question 7

What are some harmful chemical agents?

Answer 7

Acrylamide (liquid), Monosodium glutamate

Question 8

Base transition

Answer 8

Change to a base of similar shape (pyrimidine -> pyrimidine, purine -> purine)

Question 9

Base transversions

Answer 9

Change to a base of dissimilar shape (pyrimidine <-> purine)

Question 10

Depurination

Answer 10

Breakage of glycosidic bond results in AP site

Question 11

Deamination

Answer 11

Detaches NH2 from bases (again w water). C -> U, for example

Question 12

Pyrimidine dimers

Answer 12

UV radiation causing covalent linkage between T-T.

Question 13

Direct repair

Answer 13

Ex: photolyase to directly repair pyrimidine dimers by using light to break covalent bonds between adjacent pyrimidines.

Question 14

Nucleotide excision repair

Answer 14

Multi enzyme complex recognize bulky dimers (caused by UV) -> cut them out. UvrA-D, DNA pol I, ligase. Can also be done during transcription, RNA pol encounters lesion and falls off and NER is begun w same enzymes

Question 15

UvrA

Answer 15

UvrAB in complex finds bulky dimers and binds to site of distortion

Question 16

UvrB

Answer 16

UvrAB in complex finds bulky dimers and binds to site of distortion. Recruits UvrC

Question 17

UvrC

Answer 17

Flanks UvrB, cleave

Question 18

UvrD

Answer 18

Helicase! In both NER and mismatch repair

Question 19

Base excision repair

Answer 19

Glycosylases specific for each base that is not allowed in DNA seq., removes them and creates AP site. Glycosylases, AP endo+exonucleases, DNA pol, ligase

Question 20

How is damaged bases found in the DNA? (BER)

Answer 20

Flips out (no hydrogen bonds), recognized by glycosylases

Question 21

How is strand directed mismatch repair done in eukaryotes?

Answer 21

New strand have unligated nicks on them (because of the removed RNA primers) -> works as distinction

Question 22

Mismatch repair in prokaryotes

Answer 22

Dam methylase, mut-proteins involved, UvrD helicase, DNA pol III and ligase

Question 23

MutS

Answer 23

ATPase. Scans for mismatches. When it stops, binds ATP, causes conformational change that recruits MutL and MutH

Question 24

MutL

Answer 24

Recognises methylated GATC sequence, directs MutH

Question 25

MutH

Answer 25

Creates nick at hemimethylated GATC when contacted by MutL. Exonuclease activity in 5'->3' direction. Breaks down strand up until mismatch

Question 26

Pol IV & V

Answer 26

Can replicate past lesions (just adds something). Low processivity.

Question 27

Holliday model

Answer 27

A model of resolving SSB in two duplexes using homologous recombination, where there is only breaks on one strand?Involves strand invasion, branch migration, and resolution.

Question 28

Possible resolutions of Holliday model

Answer 28

Crossover product; all strands exchange information. The two uncut strands are also cut and recombined. Non-crossover product; small patch of new DNA in each duplex. No crossover between parent strands.

Question 29

RecBCD pathway

Answer 29

Pathway to resolve double stranded break in a single duplex. Involves RecA, RuvA-C. 3' ssDNA tail is generated and then ssDNA overhang. Strand invasion with this overhang.

Question 30

RecBCD

Answer 30

Exonuclease that recognize blunt ends, also helicase activity. Unwinds and breaks down. At chi-site, 5' -> 3' continues but 3' -> 5' stops.

Question 31

RecA

Answer 31

allosterically binds to ssDNA in RebBCD pathway. Two binding sites; primary for ssDNA and secondary for dsDNA that also searchs for homology in another duplex. Facilitates strand invasion

Question 32

RuvA

Answer 32

Binds to Holliday junction, recruits RuvB-duplex

Question 33

RuvB

Answer 33

ATPase, drive branch migration by hydrolysis

Question 34

RuvAB

Answer 34

catalyze branch migration

Question 35

RuvC

Answer 35

Endonuclease; resolves Holliday junctions in RecBCD pathway. Has some seq. specificity to ensure it doesnt just cleave Holliday junctions as soon as they form

Question 36

Possible resolution in RecBCD pathway

Answer 36

Patch; no crossover between duplexes, if both are cut in the same place Splice; crossover between duplexes, if they are cut on different places

Question 37

DNA microsatellites

Answer 37

Mutation-prone sequences of di-tetranucleotide sequences, causing slippage of DNA machinery

Question 38

Deamination of cytosine

Answer 38

Happens spontaneously. Creates uracil

Question 39

Alkylation of guanine

Answer 39

Results in O6-methylguanine. Mispairs with thymine.

Question 40

Oxidation of guanine

Answer 40

Caused by radiation or free radicals. Generates oxoG. Can basepair with both A and C

Question 41

Recombinases

Answer 41

Uses side chains (serine/tyrosine) to attack and cleave phosphodiester bonds, cleave all 4 strands and exchange information between duplexes. Then seals nicks themselves.

Question 42

Eukaryotes homologs of RecA

Answer 42

Rad51 and Dmc1

Question 43

Which protein introduces DSB in prokaryotes and eukaryotes?

Answer 43

none in bacteria. Spo11 in eukaryotes

Question 44

RecB

Answer 44

Helicase, exonuclease. Also makes sure RecA and not SSB coats ssDNA.

Question 45

RecD

Answer 45

Helicase, exonuclease

Question 46

RecC

Answer 46

Recognition of Chi-site