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Year 2 - MCD - Cancer > 5: DNA Damage and Repair (27.01.2020) > Flashcards

5: DNA Damage and Repair (27.01.2020) Flashcards

1
Q

What are the types of things that cause DNA damage?

A
  • deamination
  • chemical modification
  • photodamage
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2
Q

Deamination of the DNA molecule

A
  • the amino groups on the DNA molecule are somewhat unstable, they can be converted to veto groups (-NH2 -> =O)
  • a lot of the bases contain nitrogen
  • deamination reactions, e.g. cytosine -> uracil, can occur
  • Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.
  • this changes occur daily but the body is able to fix them
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3
Q

Give examples of deamination reactions

A
  • Cytosine -> Uracil (the only difference between those amino acids is the NH2 group)
  • Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.
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4
Q

Chemical modification of the DNA molecule

A
  • nucleic acid bases are susceptible to numerous modifications by a wide variety of chemical agents
  • e.g oxidation via highly reactive oxygen species
  • e.g. alkylation
  • e.g. methylation
  • e.g. adduction of larger molecules -> adducts
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5
Q

When are hyper-reactive oxygen species generated?

A
  • For example, several types of hyper-reactive oxygen (singlet oxygen, peroxide radicals, hydrogen peroxide and hydroxyl radicals) are generated as byproducts during normal oxidative metabolism.
  • Hyper-reactive oxygen species are also generated by ionizing radiation (X-rays, gamma rays).
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6
Q

How can environmental chemicals cause DNA damage?

A
  • Many environmental chemicals, including “natural” ones (frequently in the food we eat) can modify DNA bases, frequently by…
  • addition of a methyl
  • or other alkyl group (alkylation).
  • In addition, normal metabolism frequently leads to alkylation.
  • Addition of larger molecules defines “adducts”.
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7
Q

Photodamage of the DNA molecule

A
  • UV light is absorbed by NA-bases
  • there is an influx of energy
  • the most common photoproduct is bond formation between 2 adjacent pyrimidines within one strand (e.g. thymine dimers)
  • > intra DNA damage
  • UV light, radiation
  • occurs predominantly in the skin
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8
Q

What are the types of DNA damage? (on the molecule itself)

A
  • base pair mismatch (wrong pairing, usually results in a “bulge”)
  • thymine dimers (distortion of the helix)
  • gap (a lot of nicks together)
  • nick (break in the DNA phosphodiester bond)
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9
Q

Specifically what things in our surroundings can cause DNA damage and why is it important?

A

Chemicals (carcinogens)

  • dietary
  • lifestyle
  • environmental
  • occupational
  • medical
  • endogenous

Radiation

  • ionising
  • solar
  • cosmic

Important because:

  • DNA damage can lead to mutation
  • Mutation may lead to cancer
  • Damaging DNA is an important strategy in cancer therapy
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10
Q

What is an example of radiation causing DNA damage?

A

More cancer in flight crews compared to the general population (more frequent cosmic radiation exposure)

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11
Q

DNA daamage by carcinogens

A
  • DNA adducts and alkylation
  • base dimers and chemical cross links
  • double and single strand breaks
  • base hydroxylations and abasic sites formed

=> these are the predominant mutations you see

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12
Q

Abasc sites in DNA

A
  • base destroyed but the DNA structure is okay.

- due to damage

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13
Q

Polycyclic Aromatic Hydrocarbons as carcinogens

A
  • Common environmental pollutants
  • Formed from combustion of fossil fuels
  • Formed from combustion of tobacco
  • molecules with many aromatic rings
  • Can also have substituents e.g methyl groups plus N or S in the multiple rings
  • subject to drug metabolism
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14
Q

Mammalian metabolism

A

Phase I

  • addition of functional groups
  • e.g. oxidations, reductions, hydrolysis
  • mainly cytochrome p450-mediated

Phase II

  • conjugation of Phase I functional groups
  • e.g. sulphation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation
  • Generates polar (water soluble) metabolites.
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15
Q

Two step epoxidation of B[a]P

A
  • benzo [a]pyrene ➡️(via p450) B[a]p-7,8-oxide ➡️ (via EH=epoxide hydrolase) B[a]p-7,8-dihydrodiol ➡️ (via p450) B[a]p other molecule ➡️ DNA adducts
  • itself it is not carcinogenic but the metabolites are!
  • p450 and EH !!
  • In tar, tobacco smoke and grilled meat
  • associated with e.g. colon cancer
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16
Q

Epoxidation of aflatoxin B1

A
  • Formed by Aspergillus flavus mould
  • Common on poorly stored grains and peanuts
  • Aflatoxin B1 is a potent human liver carcinogen, especially in Africa and Far-East
  • p450!
  • not carcinogenic itself but after metabolism with p450 it becomes a very active epoxide
  • can react with Guanine at the N7 position -> adduct formation of guanine
  • there is a link to hepatocellulr carcinoma
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17
Q

Metabolism of 2-naphthylamine

A
  • Past components of dye-stuffs
  • Include 2-naphthylamine and benzidine
  • Potent human bladder carcinogens
  • not a carcinogen itself
  • incapable of causing cancer before it gets to the bladder (pH change)
  • CYP1A2, glucuronyl transferase, pH change
  • 2-naphthylamine ➡️ Glucuronyl ➡️ very active electrophile in the urine
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18
Q

Ioninsing radiation

A
  • there are direct and indirect effect:
    • D: interact with the bases, activation of bases, increased chemical reactivity (Possess unpaired electrons
      electrophilic and therefore seek out electron-rich DNA)
    • I: formation of superoxide radicals (O2•) and hydroxyl radicals (HO•) that can cause damage = generation of free radical sin the cells
  • causes nicks in DNA
  • e.g. higher risk of cancer in flight crews (not much ionising radiation in our normal surrounding)
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19
Q

Solar (UV) radiation as a carcinogen

A
  • Pyrimidine (thymine) dimers

- Skin cancer

20
Q

Oxygen Free Radical Attack on DNA

A
  • Double and single strand breaks
  • Apurinic & apyrimidinic sites (you lose the base structure)
  • Base modifications
    • ring-opened guanine & adenine
    • thymine & cytosine glycols
    • 8-hydroxyadenine & 8-hydroxyguanine (mutagenic) -> purines -> chemically reactive and can undergo adduct formation
21
Q

Estimated rates of endogenous damage and repair

A
  • Much more repair events than damage events!
  • In the normal situation it would appear that human cells have plenty of spare capacity to deal with both endogenous and exogenous damage but errors creep in especially with increasing age
  • The greater the persistence of damage then the greater the chance of a mutagenic event
22
Q

What is the role of p53 in dealing with cellular stress?

A
  • activation of DNA repair pathways by activation of p53
  • system that allows response to DNA damage by changing gene expression leading to activation of DNA repair pathways (eukaryotic organisms) -> underpinned by p53 in higher organisms
  • can respond to a wide variety insults to cells
  • if the damage is too severe, p53 can program the cell to undergo apoptosis.
  • p53 is a TS gene
23
Q

What are some of the cellular insults that p53 responds to?

A

Relevant here: affecting DNA replication

  • DNA replication stress
  • Double strand breaks
  • mitotic apparatus dysfunction
  • … there are more.
24
Q

How Is p53 activated/deactivated?

A
  • activated in response to cellular insults/stress
  • kept inactive by MDM2 (bound to it)
  • MDM2 is lost in the activation of p53
  • p53 is a TF -> activates gene transcriptional pathways to respond to the damage incl. DNA repair
25
Q

What activates DNA repair pathways?

A

p53 (it is a TF)

26
Q

What are the 4 main types of DNA repair?

A
  • Direct reversal of DNA damage
  • Base excision repair (mainly for apurinic/apyrimidinic damage)
  • Nucleotide excision repair (mainly for bulky DNA adducts)
  • During- or post-replication repair (MMR or recombination repair)

> 130 genes involved in repair mechanisms in mammalian cells

27
Q

Direct DNA repair

A

Direct Repair involves the reversal or simple removal of the damage by the use of proteins which carry out specific enzymatic reactions

  1. Photolyases: recognise thymine dimers, photolysis are activated by light (not UV, normal wavelength), excise dimer, the repair takes place by polymerase
    This is usually in the skin where this sort of damage occurs
  2. O6 methylguanine-DNA methyltransferase (MGMT): directly reverses some simple alkylation adducts; recognises some basis with specific methylation sites, removes the methyl group from the methylated base and restore the base identity;
    They are suicide enzymes so they are destroyed after their work is done.
28
Q

DNA Mismatch Repair

A
  • MMR involves scrutinisation of DNA for apposed bases that do not pair properly.
  • repairing erroneous insertion, deletion, and mis-incorporation of normal bases that can arise during DNA replication- failures to maintain normal Watson-Crick base pairing
  • Recognition of a mismatch requires several different proteins including one encoded by MSH2.
  • Cutting the mismatch out also requires several proteins, including one encoded by MLH1.
  • mismatch recognition and strand discrimination -> strand excision (exonuclease)-> strand resynthesis (DNA polymerase)
  • Mismatches that arise during replication are corrected by comparing the old and new strands [proof-reading].
  • Preference for newly synthesized strand.
  • Other systems deal with mismatches generated by base conversions, such as those which result from deamination.
29
Q

What happens when you inactivate the human MMR systems?

A
  • Inactivation of the human mismatch repair system confers a large increase in spontaneous mutability and a strong predisposition to tumor development.
  • Mismatch repair provides several genetic stabilization functions: it corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination.
  • Mutations in either of these genes predisposes the person to an inherited form of colon cancer.
  • So these genes qualify as tumor suppressor genes.
30
Q

How does the MMR system know which one is the incorrect nucleotide?

A
  • In E. coli, certain adenines become methylated shortly after the new strand of DNA has been synthesized.
  • The MMR system works more rapidly, and if it detects a mismatch, it assumes that the nucleotide on the already-methylated (parental) strand is the correct one and removes the nucleotide on the freshly-synthesized daughter strand.
  • How such recognition occurs in mammals is not yet known.
31
Q

Short version of DNA mismatch repair

A
  • recognition of mismatch
  • MLH and MSH are responsible for cutting and recognition respectively
  • exonuclease removes DNA strand (more than just the one base)
  • DNA polymerase resynthesises the strand

this can only occur DURING replication

32
Q

What are the types of excision repair?

A
  • NER

- BER

33
Q

Base excision repair (BER)

A
  • mutagen causes damage to the base without damaging the phosphodiester backbone
  • DNA glycolysis remove the base without damaging the backbone, you end up with an apuric situation
  • AP endonuclease cuts open the strand
  • DNA polymerase comes along and puts the right base back in
  • DNA ligase closes the gap on the pde-bond

=> important in removing SMALL adducts

34
Q

Nucleotide excision repair (NER)

A
  • mutagen causes mutation with additional damage to the DNA (e.f. big adduct group or adduct that caused more local damage)
  • ENDOLUCLEASE: makes nicks around where the damage us
  • HELICASE: removes the patch of DNA including the phosphodiester bond.
  • DNA POLUYMERASE: fills gap
  • LIGASE: seals it up
35
Q

What are some human genetic disorders involving NER?

A
  • Xeroderma Pigmentosum
  • Trichothiodystrophy
  • Cockayne’s syndrome
36
Q

Xeroderma Pigmentosum (XP)

A
  • severe light sensitivity
  • severe pigmentation irregularities
  • early onset of skin cancer at high incidence
  • elevated frequency of other forms of cancer (e.g. GI)
  • frequent neurological defects
  • extreme sensitivity to light, causes skin cancers incl melanoma; there is no treatment available, you manage it by limiting exposure to sunlight e.g. trial of making children active at night.
37
Q

Trichothiodystrophy

A
  • sulphur deficient brittle hair
  • facial abnormalities
  • short stature
  • ichthyosis (fish-like scales on the skin)
  • light sensitivity in some cases
38
Q

Cockayne’s syndrome

A
  • dwarfism
  • light sensitivity in some cases
  • facial and limb abnormalities
  • neurological abnormalities
  • early death due to neurodegeneration
39
Q

DNA double strand break repair

A
  • ends are not matching; the DNA repair machinery chews up the ends to reveal more ssDNA with the hope to find matching sequences.
  • Ku proteins are a part of it
  • desperate attempt to get some double stranded DNA back from DNA fragments
  • recombinational DNA
40
Q

What are the different fates of DNA damage?

A
  • efficient repair -> normal cell
  • incorrect repair, altered 1* sequence -> a) carcinogenesis (oncogenes, TS genes) or b) aberrant proteins
  • Apoptosis -> cell death (p53)
41
Q

How does cancer chemotherapy work?

A
  • you want to damage the DNA so much that the cell will undergo apoptosis (e.g. alkylating agents)
42
Q

Testing for carcinogens tests

A
  • structural analysis (can tell how likely it is that a chemical with a certain structure is in fact a carcinogen)
  • in vitro bacterial gene mutation assay (e.g. Ames test)
  • in vitro mammalian cell assay (look for abberrant chromosomes or micronucleus assay)
  • in vivo mammalian cell assay
  • investigative in vivo cell assays
43
Q

What is the Ames test?

A
  • used in the early steps of testing for carcinogens
  • bacteria in a Petri dish that do not synthesise histidine
  • plus add rat liver enzymes (p450 enzymes for Ph1 and Ph2 metabolism)
  • if the bacteria acquire the ability to make histidine, they will form colonies and this mean a mutation has occurred
  • there is a negative control (no carcinogen) and a positive control (known carcinogen, will grow colonies)
  • the results are quantifiable as you can count the colonies
44
Q 
Detecting DNA Damage in Mammalian Cells
Chromosomal Abberrations (in vitro)
A
  • grow cells with the carcinogen and the rat liver enzymes (p450 to allow for Ph1 and Ph2 metabolism)
  • see if any chromosomal damage has occurred, look for aberrant chromosomes.
45
Q

In vitro micronucleus assay

A
  • if DNA damage is very severe the DNA is divided off into a micronucleus
  • Cells treated with chemical and allowed to divide
  • Binucleate cells assessed for presence of micronuclei
  • Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss
46
Q

Murine Bone Marrow Micronucleus Assay

A
  • usually done in rats
  • you feed them the carcinogen and see the effects of it without looking for tumours
  • look at BM: see how often you detect micronuclei in polychromatic erythrocytes (which shouldn’t have any at all because nuclei are lost earlier in RBCs)

Year 2 - MCD - Cancer (15 decks)

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