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3382 > DNA Damage & Repair > Flashcards

DNA Damage & Repair Flashcards

1
Q

What is DNA damage?

A

chemical alteration to DNA that does NOT always lead to a mutation

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2
Q

What is a mutation?

A

permanent change in a bp

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3
Q

error-prone DNA repair is advantageous because

A

promotes genetic variation in response to environmental challenges (natural selection)

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4
Q

What are the 3 cause of DNA damage?

A
  1. environmental agent: radiation/heat
  2. chemical agents: benzenes
  3. spontaneous nutations (errors in DNA repl/repair)
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5
Q

What are the types of chemical damage that DNA is susceptible to?

A
  • oxidation
  • hydrolysis (of C to U)
  • methylation (at nitrogens of base)
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6
Q

What is the effect of environmental agents on DNA?

A
  • UV light
  • cyclobutyl ring/thymine dimer on adjacent T-T on same strand
  • distorts local structure
  • interferes with transcription and DNA rep
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7
Q

Types of chemical mutagens?

A
  1. base analog
  2. base altering chemicals (HNO2)
  3. intercalating agent
  4. DNA structure altering agents
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8
Q

What is the effect of chemical mutagens?

A

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

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9
Q

What is spontaneous mutations?

A
  • errors in DNA rep not fixed by DNAP proofreading
  • called mismatches: nucleotide inserted that doesn’t bp w/ template
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10
Q

Why do spontaneous mutations need to be fixed before next replication round?

A

DNA can’t tell which is parent strand

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11
Q

Why is the rate of natural mutation so low (euk. cells: 10^-11 mutations/bp)?

A
  • polymerase base selectivity
  • 3’-5’ exonucleave activity proofreading
  • mismatch correction
  • DNA damage repair
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12
Q

Biological consequence of silent mutations? Where do silent mutations occur?

A

no cons even though DNA seq is mutated
- non-coding DNA region
- synonymous AA change

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13
Q

Biological consequence of deleterious mutations? Where do deleterious mutations occur?

A

mutation impacts function
- regulatory DNA seq (promoter)
- RNA ss (2º structure that makes RBS inaccessible)
- coding region

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14
Q

What are the types of mutations in coding regions?

A
  1. synonymous mutation: doesn’t change codon identity
  2. nonsynonymous: does change codon identity
  3. nonsense mutation: create stop codon
  4. readthrough: mutation makes a stop codon into an AA codon
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15
Q

What are the DNA repair pathways?

A

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

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16
Q

How was photolyase discovered to be part of E.coli direct repair?

A
  1. E.coli strain with DNA repair mechs KO
  2. add plasmid w/ photolyase gene
  3. expose E.coli to UV light
  4. keeps some cells in light and some in dark
  5. measure survival

UV light treatment = cell death
light exposure after promotes cell survival

17
Q

What were the results/interpretation of the photolyase experiment?

A
  • DNA repair is essential
  • DNA photolyase gene required for repair of UV induced DNA damage
  • light is essential for repair processes
18
Q

Why can’t mammalian cells do direct repair? How do they fix T-T dimers?

A
  • don’t depend on white light
  • use nucleotide excision repair for dimers
19
Q

What is the DNA photolyase DR reaction pathway

only in E.coli

A
  1. T=T dimer formed with UV
  2. recognize kink in bb so DNA photolyase binds to dimer
  3. white light causes FADH- to transfer electrons to ring, which flips out to expose to photolyase active site and break ring
  4. repaired DNA released and electrons return to FADH
20
Q

What are the types of excision repair?

A

BER: repairs small, non-helical distortions from DNA (removes only base)
NER: repairs bulky, helical-distorting DNA damage (remove whole nucleotide)

21
Q

How does Base Excision Repair work?

how does uracil DNA glycosylase work

A
  1. DNA glycosylase removes base (phosphate and sugar remain) making an AP site
  2. AP endonuclease breaks bb to make single nucleotide gap
  3. DNAP + ligase

uracil DNA glycosylase flips uracil out into the active site

22
Q

How does Nucleotide Excision Repair work?

A
  1. helicase separates strands
  2. endonuclease cleaves to remove a few bases
  3. polymerase + ligase

DIFFERENT FOR E.COL VS EUKARYOTES

23
Q

How does E.Coli NER work?

A
  1. T=T dimer recognized by UvrA homodimer which sees kink
  2. UvrA recruits UvrB
  3. this forms a DNA complex at the kink and flips DNA to expose dimer
  4. UvrC enconuclease makes 5’-3’ cut on either side of T=T
  5. UvrD helicase displaces UvrC and DNA fragment
  6. DNAP + ligase
24
Q

Difference between E.coli and Eukaryotic NER?

A

E.coli doesn’t have ssBP and has helicase used later on while eukaryotes have it 1st

25
Q

How does Eukaryotes NER work?

A
  1. uses XP proteins
  2. XPC recruits other proteins
  3. RPA ssBP exposes one strand (ss)
  4. TFIIH: helicase activity
  5. DNAP + ligase
26
Q

What is mismatch repair?

A

corrects errors in DNA rep due to DNAP

mismatch: wrong base put by DNAP

27
Q

Why is BER and NER not suitable for mismatch repair?

A

mismatch is not DNA damage
BER and NER recognize abnormal structure which is not indicated in a mismatch

28
Q

What was results from the in vitro assay for mismatch repair activity?

A
  • ATP dependent process
  • NAD+ independent
  • requires dNTP for better result from nuclear extract, not exogenous
  • methylation required ONLY on strand opposite to mismatch
  • if methylated on none or both = no repair
29
Q

What are the steps of the Mismatch Repair Pathway?

A
  1. MutS recognizes a difference in DNA structure
  2. MutS recruits MutL
  3. MutL and ATP creates liip structure to look for hemimethylated site
  4. MutH endonuclease cleaves methylated site on new strand
  5. UvrD helicase displaces new strand
  6. exonucleave degrades strand
  7. DNAP + ligase

UvrD is used in mismatch repair and E.coli NER

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