DNA Damage & Repair Flashcards
What is DNA damage?
chemical alteration to DNA that does NOT always lead to a mutation
What is a mutation?
permanent change in a bp
error-prone DNA repair is advantageous because
promotes genetic variation in response to environmental challenges (natural selection)
What are the 3 cause of DNA damage?
- environmental agent: radiation/heat
- chemical agents: benzenes
- spontaneous nutations (errors in DNA repl/repair)
What are the types of chemical damage that DNA is susceptible to?
- oxidation
- hydrolysis (of C to U)
- methylation (at nitrogens of base)
What is the effect of environmental agents on DNA?
- UV light
- cyclobutyl ring/thymine dimer on adjacent T-T on same strand
- distorts local structure
- interferes with transcription and DNA rep
Types of chemical mutagens?
- base analog
- base altering chemicals (HNO2)
- intercalating agent
- DNA structure altering agents
What is the effect of chemical mutagens?
point mutations: mispairing
- deamination of C to U (CG -> UA -> TA)
idel: ORF shift
- acridine orange intercalating agent makes atypical spacing between nucleotide, makes DNAP mistakes
What is spontaneous mutations?
- errors in DNA rep not fixed by DNAP proofreading
- called mismatches: nucleotide inserted that doesn’t bp w/ template
Why do spontaneous mutations need to be fixed before next replication round?
DNA can’t tell which is parent strand
Why is the rate of natural mutation so low (euk. cells: 10^-11 mutations/bp)?
- polymerase base selectivity
- 3’-5’ exonucleave activity proofreading
- mismatch correction
- DNA damage repair
Biological consequence of silent mutations? Where do silent mutations occur?
no cons even though DNA seq is mutated
- non-coding DNA region
- synonymous AA change
Biological consequence of deleterious mutations? Where do deleterious mutations occur?
mutation impacts function
- regulatory DNA seq (promoter)
- RNA ss (2º structure that makes RBS inaccessible)
- coding region
What are the types of mutations in coding regions?
- synonymous mutation: doesn’t change codon identity
- nonsynonymous: does change codon identity
- nonsense mutation: create stop codon
- readthrough: mutation makes a stop codon into an AA codon
What are the DNA repair pathways?
direct repair systems: act directly on damaged nucleotides
excision repair: excision of a base or segment of damaged DNA
mismatch repair: corrects errors of replication
NHEJ: used to replain ds breaks
How was photolyase discovered to be part of E.coli direct repair?
- E.coli strain with DNA repair mechs KO
- add plasmid w/ photolyase gene
- expose E.coli to UV light
- keeps some cells in light and some in dark
- measure survival
UV light treatment = cell death
light exposure after promotes cell survival
What were the results/interpretation of the photolyase experiment?
- DNA repair is essential
- DNA photolyase gene required for repair of UV induced DNA damage
- light is essential for repair processes
Why can’t mammalian cells do direct repair? How do they fix T-T dimers?
- don’t depend on white light
- use nucleotide excision repair for dimers
What is the DNA photolyase DR reaction pathway
only in E.coli
- T=T dimer formed with UV
- recognize kink in bb so DNA photolyase binds to dimer
- white light causes FADH- to transfer electrons to ring, which flips out to expose to photolyase active site and break ring
- repaired DNA released and electrons return to FADH
What are the types of excision repair?
BER: repairs small, non-helical distortions from DNA (removes only base)
NER: repairs bulky, helical-distorting DNA damage (remove whole nucleotide)
How does Base Excision Repair work?
how does uracil DNA glycosylase work
- DNA glycosylase removes base (phosphate and sugar remain) making an AP site
- AP endonuclease breaks bb to make single nucleotide gap
- DNAP + ligase
uracil DNA glycosylase flips uracil out into the active site
How does Nucleotide Excision Repair work?
- helicase separates strands
- endonuclease cleaves to remove a few bases
- polymerase + ligase
DIFFERENT FOR E.COL VS EUKARYOTES
How does E.Coli NER work?
- T=T dimer recognized by UvrA homodimer which sees kink
- UvrA recruits UvrB
- this forms a DNA complex at the kink and flips DNA to expose dimer
- UvrC enconuclease makes 5’-3’ cut on either side of T=T
- UvrD helicase displaces UvrC and DNA fragment
- DNAP + ligase
Difference between E.coli and Eukaryotic NER?
E.coli doesn’t have ssBP and has helicase used later on while eukaryotes have it 1st
