lecture 5 - dna recombination, damage and repair

Question 1

what is general recombination

Answer 1

part of meiosis that results in crossing over of chromosomes

Question 2

what is the holliday model

Answer 2

proposed in 1964 to explain formation of heteroduplexes and gene conversion (has been replaced but some details still hold)

Question 3

explain steps in holliday model

Answer 3

1. nicks are made in one strand of both chromosomes
2. 3’ ends of cut strands cross and join 5’ end of homologous strand
3. branch migration forms heteroduplex region
4. rotation at crossover gives isomeric hollidaystructur
5. makes cuts in two places to give either heteroduplexes and recombinants or heteroduplexes and no recombinants

Question 4

why may the holliday model not be correct

Answer 4

because there are breaks in both strands of one homolog to start recombination

Question 5

what is the more likely method of recombination

Answer 5

double stranded break

Question 6

how does double stranded break recombination work

Answer 6

1. generation of double stranded break in dna
2. processing of ends gives 3’ overhand
3. homologous dna is recruited for repair
4. strand displacement results in d loop formation so there is 3’ single stranded invasion
5. 3’ ends extends using homologous dna as template
6. double holliday junction occurs
7. this junction is resolved

Question 7

what does RecA do in recombination

Answer 7

binds to single stranded DNA and promotes invasion of double stranded heliz

Question 8

what does RuvA/B do in recombination

Answer 8

promote strand/branch migration

Question 9

what does Ruv C do in dna recombniation

Answer 9

resolve holliday structure by cleaving branches and the ligation

Question 10

what are the types of point mutations and what are they

Answer 10

transition –> purine replace by purine, pyrimidine replace by prymidine
transversion –> purine replace by pyrimidine or vice cersa
missense –> amino acid substitution
nonsense –> introduces a stop codon

Question 11

what are the types of dna mutations

Answer 11

point mutations, insertions/deletions, double stranded breaks

Question 12

how do endogenous mutations occur

Answer 12

replication errors (issues with DNA polymerase/replication) or metabolic byproducts (reactive oxygen species)

Question 13

how do exogenous mutations occur

Answer 13

mutagenesis (dna damage by chemical agents), carcinogenesis (mutations that alter cellular metabolism), ionizing and non-ionizing radiation (cause pyrimidine dimers and DSBs)

Question 14

what can cause transitions/transversions

Answer 14

oxigation, oxidative deamination and aklkyation (8 oxo guanine can bind with adenine, with HNO2 then cytonise can become uracil and bind with adenine and adenine can become hypoxanthine which binds with cytosine)

Question 15

what can UV do to DNA

Answer 15

cause pyrimidine dimer which bonds 2 pyrimidines together and will cause kink in helix

Question 16

what are the DNA repair pathways

Answer 16

direct reversal, nucleotide excision repair, base excision repair, mismatch repair, non-homologous end joining, homology directed repair

Question 17

how does direct reversal work

Answer 17

use energy from sunlight to activate photolyase enzyme that repairs pyrimidine dimers (not possible in mammals)

Question 18

how does base excision repair (BER) work

Answer 18

DNA glycosylase removes the damaged base leaving an AP site. AP endonuclease breaks strands and removes some bases. DNA pol I and DNA ligase fix this gap

Question 19

what is the most common pathyway for DNA repair

Answer 19

BER

Question 20

what is nucleotide excision repair used for (NER)

Answer 20

bulky repairs

Question 21

how does NER work

Answer 21

UrvA,B,C cleaves at 7th phosphodiester bond 5’ to damage and 3rd/4th bond to 3’ damage and then bases are replace by DNA pol

Question 22

how are most replication errors/short insertions deletions corrected

Answer 22

mismatch repair (MMR)

Question 23

how does MMR work

Answer 23

the parent DNA strand is methylated and the MutS recognizes mismatch. MutL makes a loop in the DNA which attracts MutH to nick 5’ base of unmethylated strand. The bases are removed and fixed by DNA pol/ligase. The new strand is methylated

Question 24

what is the most common pathway in humans to repair DSB

Answer 24

NHEJ

Question 25

what pathways repair DSBs

Answer 25

NHEJ and HDR

Question 26

how does NHEJ work

Answer 26

works almost immediately. The ends of the DNA are jammed together and ligated. Ku70.Ku80 recognize dSB

Question 27

does NHEJ give perfect chromosomes

Answer 27

no bc usually there are loss/gains of bases at the DSB site

Question 28

how does homology directed repair work

Answer 28

only possible when homologous region of DNA is available. MRN complex recognize break and the ends are resected to have single stranded regions. The SS regions invade to form holliday junctions and the branches migrate. holliday structures resolved by resolvase.

Question 29

what are illnesses/diseases from problems in repair system

Answer 29

xeroderma pigmentosum –> defect in NER
cockayne –> defect in NER and other pathways
human nonpolposis colorectal cancer –> defect in MMR
hereditary brest cancer –> defects in BRCA1 or 2 (part of NHEJ pathway)