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Genes and Genomics > DNA Damage & Repair > Flashcards

DNA Damage & Repair Flashcards

1
Q

Effects of Damage

A
  • block replication or transcription via shape changes

- causes alterations to genetic code

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2
Q

2 causes of damage

A
  1. chemical alteration to DNA (exogenous ie. environmental or endogenous ie. internally generated damaging agents like hydroxyl radicals)
  2. spontaneous damage (deamination or depurination)
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3
Q

Pyrimidine Dimers

A
  • exogenous UV mutation
  • 2 adjacent pyrimidines joined by a cyclobutane ring structure
  • form new covalent bonds + DNA lesion
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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
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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
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6
Q

Deamination

A
  • adenine, cytosine, guanine
  • causes different H bonding
  • adenine deamination to hypoxanthine causes T-C pairing as it resembles G
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7
Q

Depurination

A
  • results from cleavage of bond between purine bases and deoxyribose leaving apurinic site
  • leaves reactive hydroxyl on DNA chain
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8
Q

2 Types of Repair Mechanism

A
  1. direct reversal of chemical reaction responsible for damage (bacteria)
  2. excision repair of damaged bases and replacement with newly synthesized DNA (humans)
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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

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10
Q

Mechanisms of Excision Repair

A
  1. base excision repair: removes base and leaves backbone
  2. nucleotide excision repair: removes nucleotide leaving strand gap
  3. mismatch repair: repair of post replication errors
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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
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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

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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)
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14
Q

Mismatch Repair in E. Coli

A
  • excises mismatched bases in newly replicated DNA

- methylation distinguishes parental from daughter strand

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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
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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
Q

Holliday Junction

A
  • 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
Q

RuvAB

A
  • 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
Q

Resolution of Junction

A
  • gene conversion: cutting junction horizontally

- crossover: cutting junction vertically

28
Q

RuvC

A
  • cleaves junctions
  • position of binding/cleavage delineates outcome
  • patch and splice variations
29
Q

HR Uses

A
  • variation in offspring
  • gene shuffling
  • chromosome alignment during meiotic prophase
  • horizontal gene transfer
  • DNA repair
30
Q

Recombinases

A
  • site specific recombination
  • shorter homologous DNA than HR
  • can have inversion, deletion, insertion depending on repeat orientation
31
Q

Integrases

A
  • 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
Q

Transposons

A
  • 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

Genes and Genomics (13 decks)

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