DNA Damage & Repair Flashcards
1
Q
Effects of Damage
A
- block replication or transcription via shape changes
- causes alterations to genetic code
2
Q
2 causes of damage
A
- chemical alteration to DNA (exogenous ie. environmental or endogenous ie. internally generated damaging agents like hydroxyl radicals)
- spontaneous damage (deamination or depurination)
3
Q
Pyrimidine Dimers
A
- exogenous UV mutation
- 2 adjacent pyrimidines joined by a cyclobutane ring structure
- form new covalent bonds + DNA lesion
4
Q
Alkylation
A
- exogenous mutation
- addition of methyl/ethyl groups to bases
eg alkylation of O6 position of guanine forms O6 methylguanine - affects base pairing
5
Q
Carcinogens
A
- react with DNA bases to add large bulky chemical groups to DNA molecules
- activated endogenously by reacting with cytochrome P450 enzymes that cause reactive intermediates
- causes heritable mutations and unfaithful base pairing
6
Q
Deamination
A
- adenine, cytosine, guanine
- causes different H bonding
- adenine deamination to hypoxanthine causes T-C pairing as it resembles G
7
Q
Depurination
A
- results from cleavage of bond between purine bases and deoxyribose leaving apurinic site
- leaves reactive hydroxyl on DNA chain
8
Q
2 Types of Repair Mechanism
A
- direct reversal of chemical reaction responsible for damage (bacteria)
- excision repair of damaged bases and replacement with newly synthesized DNA (humans)
9
Q
Direct Reversal
A
eg. pyrimidine dimers
- photoreactivation: direct reversal of pyrimidine dimerisation uses visible light to break cyclobutane ring
eg. alkylation
- methylation of guanine base pairs with T
- repaired by enzyme with cysteine in active site
- sulfhydryl rips off methyl to restore carbonyl original group
10
Q
Mechanisms of Excision Repair
A
- base excision repair: removes base and leaves backbone
- nucleotide excision repair: removes nucleotide leaving strand gap
- mismatch repair: repair of post replication errors
11
Q
Base excision repairs
A
- uracil formed by deamination of cytosine leads to GU mismatch
- bond to deoxyribose cleaved by uracil DNA glycosylase leaving an AP site
- AP endonuclease cleaving DNA chain and deoxyribose removed by deoxyribose-phosphodiesterase
- resulting gap filled by DNA polymerase and sealed by ligase
- incorporation of C opposite G
12
Q
Nucleotide excision repairs
A
eg. repair of thymine dimers
- damaged DNA recognised on both sides by endonucleases
- unwinding causes excision
- gap filled and sealed
13
Q
Thymine Dimer Repair (E. Coli vs eukaryotes)
A
E. Coli - 3 gene products - UvrA recognises damage, UvrB/UvrC cleave Eukaryotes - 7 repair genes in humans - Xeroderma pigmentosum (lack of repair)
14
Q
Mismatch Repair in E. Coli
A
- excises mismatched bases in newly replicated DNA
- methylation distinguishes parental from daughter strand
15
Q
MutHLS Mismatch Components
A
- requires communication between site of damage and new strand identifier
- MutS: recognition of mismatch
- MutL: binds MutS at mismatch
- ATPase activity forming DNA loop translocating along DNA looking for hemi-methylated Dam site
- MutH: endonuclease binding to MutL
- binds to MutL endonuclease cleaves unmodified strand opposite a site of hemi-methylation
- discriminates newly synthesized DNA
- presence of complex signals mismatch is present
16
Q
MutHLS Mismatch Repair
A
- nick can be either upstream or downstream of mismatch
- different exonucleases required depending on polarity
- helicase and exonuclease excise daughter strand with mismatch
- gap filled and sealed
17
Q
Mismatch Repair in Mammalian Cells
A
- newly replicated strand is distinguished from parental strand using strand breaks
- MSH complex (homologous to Mut) responsible for mismatch repair
- looking for strand breaks close to mismatch on same strand
18
Q
Non-Homologous End Joining
A
- error prone process requiring resection of ends prior to ligation
19
Q
Homologous Recombination
A
- error free
- requires homologous DNA providing new template
- template can be found at replication fork
- deals with lesions and double strand breaks
- gap repair mechanism when template is lost
- uses homology in break ends and reads template from 2nd copy (parallel chromosome) to replace missing information
20
Q
Homologous Recombination Mechanism
A
- breaking 2 homologous strands
- pairing of strand
- reforming phosphodiester bonds
- breaking other two strand and joining them
21
Q
Types of Crossover
A
Intermolecular
- single crossover: swap info between sister chromosomes
- double crossover: swap chromosome regions
Intramolecular
- direct repeats: excise intervening DNA in between repeats
- inverted repeats: flips region between repeats
22
Q
E. Coli RecBCD Pathway
A
- double strand break
- RecBCD unwinds DNA and degrades one strand in 5’-3’
- 3’ ssDNA bound by RecA to form filament
- ss RecA filament invades homologous strand: invades D loop formed within a region of homologous dsDNA/pauses and anneals via bp to homologous target
- nicking and strand exchange of homologue: primes onto template strand
- fill in and ligate
- Holiday junction
- branch migration
23
Q
Strand Invasion
A
- Rec filament formation known to be essential for strand invasion
- strand invades homologous template forming a D bubble
- RecA released from target site
- 5’ end acts as a primer for DNAP
24
Q
RecA
A
- helical nature forms triplex structure with homologous douplex of DNA
- one original strand displaced by invading strand
- ATP drives process
25
Holliday Junction
- invaded duplex nicked
- 5' end is a primer annealing to top blue strand
- gaps on strands re-sealed
- holliday junctions forms
- branch migration promotes exchange
26
RuvAB
- RuvA is a flat structure binding 4-way Holliday junction
- hydrophobic pin in the middle separates strands
- RuvB motors bind either side using ATP to translocate DNA
- fundamental unit of recombination and DNA transfer
27
Resolution of Junction
- gene conversion: cutting junction horizontally
| - crossover: cutting junction vertically
28
RuvC
- cleaves junctions
- position of binding/cleavage delineates outcome
- patch and splice variations
29
HR Uses
- variation in offspring
- gene shuffling
- chromosome alignment during meiotic prophase
- horizontal gene transfer
- DNA repair
30
Recombinases
- site specific recombination
- shorter homologous DNA than HR
- can have inversion, deletion, insertion depending on repeat orientation
31
Integrases
- insert virus genome
eg. lambda phase integrase
- site specific at att sites (attP viral and attB bacterial
- homology at O sequence
- Integrase brings together homologous sites for insertion
- Tyr is enzyme active site traps energy in high enegy intermediate that reacts with other strands
32
Transposons
- mobile genetic elements moving randomly
- sequence homology not required
- simple transposons contain genes for their jump
- complex transposons contain other genes
- found in horizontal gene transfer
