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MCD - Cancer > DNA damage and repair > Flashcards

DNA damage and repair Flashcards

1
Q

What can damage DNA?

A
  1. Chemicals (carcinogens)
    - Dietary (strongly associated w/cancer – 40%)
    - Lifestyle
    - Environmental, e.g. herbicides, pesticides (small burden)
    - Occupational
    - Medical, e.g. drugs, radiotherapy
    - Endogenous, e.g. viruses, infections, reactive O2 species produced by mitochondria
  2. Radiation
    - Ionising, e.g. X-rays
    - Solar - UV
    - Cosmic
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2
Q

How does p53 detect and respond to DNA damage?

A

Crucial TSG

  • Normally tied up with MDM2, which keeps p53 inactive
  • When released from MDM2, p53 forms a dimer that activates many pathways
  • Mild physiological stress e.g. DNA repair or growth arrest –> p53 orchestrates a transcriptional series of events and activates proteins that help repair problem
  • SEVERE stress –> p53 can activate an apoptotic pathway by directly interacting w/apoptosis proteins
  • Many different stresses can kick p53 into activation
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3
Q

What are the natural repair mechanisms for damaged DNA?

A
  1. Direct reversal of DNA damage
  2. Base excision repair
  3. Nucleotide excision repair
  4. During or post-replication repair
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4
Q

How can unrepaired DNA become fixed as a mutation?

A
  • If damage is not severe, p53 will not commit cell to apoptosis
  • p53-triggered pathways could lead to incorrect repair/altered primary sequence
  • DNA replication and cell division –> fixed mutation in daughter cells (permanent mutations)
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5
Q

How is the potential of a chemical agent to damage DNA assessed?

A

Tiered approach

  1. Evaluate structure of chemical - look for potentially reactive FGs (structural alerts)
  2. In vitro BACTERIAL gene mutation assay (AMES test w/S. typhimurium)
  3. In vitro MAMMALIAN CELL assay (chromosome aberration, micronucleus assay)
  4. In vivo MAMMALIAN assay (bone marrow micronucleus assay in mice)
  5. Investigative in vivo MAMMALIAN assays
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6
Q

How can double strand breaks cause problems?

A

Tendency for 2 bits of DNA to drift apart
Intolerable for cell
DNA repair may go wrong and introduce DNA damage

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7
Q

How can DNA adducts and alkylation cause problems?

A
  • Some chemicals tend to be metabolically activated into electrophiles
  • DNA is very rich in electrons bc of nitrogens in bases
  • Electrophiles bind to DNA and form covalent bond
  • Binding of a big bulky chemical to DNA causes problems particularly during replication bc DNA polymerase can’t recognise base bc chemical adduct gets in the way
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8
Q

If DNA damage is excessive, what usually happens?

A

p53

Apoptosis

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9
Q

If DNA damage is small, what usually happens

A

p53
Efficient repair
Normal cell

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10
Q

What happens if repair is incorrect or the primary sequence is altered after repair?

A

DNA replication and cell division
Fixed mutations in daughter cells
Transcription/translation –> aberrant proteins
Carcinogenesis if critical targets are mutated: oncogenes, TSGs

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11
Q

How does solar (UV) radiation damage DNA?

A 
UV radiation induces pyrimidine dimers
(CUT)
2 adjacent pyrimidines covalently link
Mutation can occur during DNA repair
(--> skin cancer)
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12
Q

How does ionising radiation damage DNA?

A

Can generate free radicals (O2., HO.)in cells
Good defence mechanisms but ionising radiation can overwhelm these
Induces pyrimidine dimers, strand breaks, abasic sites and modified bases in DNA

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13
Q

How is the bacterial Ames test used to assess the mutagenicity of a chemical?

A
  • Salmonella typhimurium normally used for assay
  • Incubate chemical w/preparation of rat liver enzymes (contained P450) to generate reactive chemical (chemicals often need to be metabolically activated before they are carcinogenic)
  • Bacteria genetically engineered so they can’t produce histidine
  • Bacteria require exogenous histidine to grow
  • On histidine-free media, if mutations occur in bacterial genome then bacteria acquire ability to synthesise histidine –> grow into colonies
  • The more the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine
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14
Q

How are chromosomal aberrations used to assess the mutagenicity of a chemical?

A
  • Chemicals can cause double strand breaks that leads to fragmentation of chromosomes
  • Treat mammalian cells with chemical in presence of liver s9 (to metabolically activate)
  • Look for chromosomal damage
  • If chromosomes are damaged in any way, mammalian cell has been damaged by chemical
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15
Q

How are in vitro micronucleus assays used to assess the mutagenicity of a chemical?

A
  • Cells treated w/chemical
  • Allow cell to go through 1 replication cycle
  • Binucleate cells assessed for presence of micronuclei - pieces of chromosomal material that have broken off and no longer appear in nucleus
  • Chemicals that damage DNA can generate these
  • Kinetochores of chromosomes can be stained to determine if chemical treatment caused:
    1. Clastogenicity - chromosomal breakage
    2. Aneuploidy - loss or change in no. of chromosomes
    ….both can contribute to cancer
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16
Q

How are bone marrow micronucleus assays in mice/rats used to assess the mutagenicity of a chemical?

A
  • Use pluripotent nature of bone marrow in producing blood cells
  • Treat animals w/chemical
  • Examine BM cells or peripheral erythrocytes for presence of micronuclei (indicates DNA damage)
  • Erythrocytes normally remove nucleus during development, but can’t remove micronuclei
17
Q

How can DNA damage be directly reversed?

A
  • Photolyase splits and cuts out cyclobutane pyrimidine dimers generated by UV radiation
  • Methyltransferases and alkyltransferases remove alkyl groups from bases
18
Q

How can DNA damage be repaired by base excision?

A

Mainly for apurinic-apyrimidic damage

  • 1 base missing from DNA
  • DNA glycosylase splits link between sugar and DNA base
  • AP endonuclease splits DNA strand to make gap in backbone
  • DNA repair polymerase fills in missing base
  • DNA ligase seals DNA to form intact DNA
19
Q

How can DNA damage be repaired by nucleotide excision?

A

Mainly for bulky DNA adducts

  • Xeroderma pigmentosum proteins assemble at site of damage
  • A stretch of nucleotides on either side of damage are excised
  • Endonuclease makes 2 cuts in DNA on either site of damage
  • Patches can be long (100-200 nucleotides) or short (10-20)
  • Patch of DNA is removed
  • Helicase unwinds DNA, leaving double stranded DNA with patch missing
  • DNA polymerase replaces removed bases using complementary strand as template
  • DNA ligase joins DNA up again
20
Q

How can DNA damage be repaired during or post-replication?

A
  • Mismatch repair
  • Recombinational repair
  • Check DNA to make sure it’s ok before daughter cells bud off in mitosis

MCD - Cancer (16 decks)

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