DNA Damage and Repair

Question 1

What can damage DNA?

Answer 1

CHEMICALS (carcinogens): dietary, lifestyle, environmental, occupational, medical and endogenous

RADIATION: ionising, solar and cosmic

ENDOGENOUS: mitochondria produce reactive oxygen species that may damage DNA, infection etc

Question 2

How do carcinogens cause DNA damage?

Answer 2

base dimers and chemical cross links (DNA molecules chemically linked up)

base hydroxylation
(oxidative reaction on DNA base -> requires repair - muation could occur)

abasic sites (DNA base removed during repair - missing base causes problems during replication)

single strand breaks (tropoisomerase relaxes and unwinds DNA -> access to DNA as strand is reannealed)

double strand breaks ( tendency for strands to drift apart -> DNA damage when repair goes wrong

DNA adducts and alkylation (some chemicals are electrophiles -> binds to DNA via covalent bond - during replication DNA polymerase won’t know which base to put in next)

Question 3

What happens in phase 1 of mammalian metabolism?

Answer 3

Addition of functional groups (these reactions introduce or unmask functional groups) e.g. oxidations, reductions, hydrolysis

• Mainly cytochrome p450-mediated (oxidation) – broad substrate specificity and oxidise chemicals

Question 4

What happens in phase 2 of mammalian metabolism?

Answer 4

conjugation of Phase I functional groups (the functional groups are used) e.g. sulphation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation

• Generates polar (water soluble) metabolites by adding a polar endogenous group - take something lipophilic and make it more polar so we can excrete it

Question 5

How are polycyclic aromatic hyrdocarbons formed?

Answer 5

combustion of fossil fuels and tobacco

Question 6

What is benzo[a]pyrene metabolised to?

Answer 6

It is oxidised by CYP450, to produce an epoxide/oxide. This is reactive and unstable (and potentially damaging). - Epoxide hydrolase metabolises this molecule, to form dihydrodiol. This is harmless.

Dihydrodiol is also a subrate for CYP450 - oxidation to form a diol epoxide - not stable at all -> forms DNA adducts -> mutations

Question 7

What forms aflatoxin B1?

Answer 7

aspergillus flavous mould

- common on poorly stored grains/peanuts

Question 8

How does aflatoxin B1 cause mutations in DNA?

Answer 8

Aflatoxin B1 is oxidised by P450 -> aflatoxin B1-2,3-epoxide (VERY REACTIVE)

• reacts with the N7-position of guanine to form big, bulky, chemical DNA adducts - DNA in the cell is now read as damaged -> fixed inappropriately -> mutations

Question 9

Why does aflatoxin mainly target the liver?

Answer 9

mainly activated by P450 found in liver (different P450 found in lots of differnt tissues -> PAH cause cancer in lots of places)

Question 10

What type of cancer does napthylamine cause?

Answer 10

bladder

Question 11

How does napthylamine cause cancer?

Answer 11

2-naphthylamine is a substrate for CYP450 -> converts the amino group to form a hydroxylamine (reactive) -> glucuronidated (detoxified) in the liver by glucuronyl transferase -> excreted by the liver and it goes into the bladder and mixes with the urine

• Urine is ACIDIC, and, under acidic conditions, the glucuronides are hydrolysed -> release the hydroxylamine derivative

In the acidic conditions, the molecule rearranges to form a positively charged nitrogen (nitrenium ion - electrophile) -> binds to the DNA and forms adducts

• The bladder isn’t as capable of detoxifying the hydroxylamine derivative as the liver

Question 12

How does UV light cause skin cancer?

Answer 12

UV radiation can lead to the formation of Pyrimidine Dimers

• Pyrimidines = cytosine + uracil + thymine (CUT)

2 pyrimidines next to each other, in the presence of UV radiation, they can covalently link - cell tries to repair this, but in doing so, a mutation is introduced

Question 13

How does ionising radiation cause cancer?

Answer 13

all ionising radiation generates free radicals in cells (includes oxygen free radicals) - very reactive

Free radicals possess unpaired electrons: electrophilic and therefore seek out electron-rich DNA

• Super Oxide Radical – molecule of oxygen that has an extra electron so it is very reactive
• Hydroxyl Radical – hydroxyl group that has grabbed an extra electron
• This is even more reactive than the super oxide radical (very electrophilic and DNA is electron-rich)

Question 14

How do free radicals cause mutations?

Answer 14

Double stranded breaks have to be re-annealed and rebuilt, which can introduce mutations -> generate apurinic and apyrimidic sites – base stripped out of the DNA

• The base has been oxidised by an oxygen free radical and the DNA repair enzymes cut out the base itself -> leaves the sugar-phosphate backbone in tact so there are gaps (abasic sites)

Also introduces base modifications:

• Ring-opened guanine + adenine
• Thymine + cytosine glycols
• 8-hydroxyadenine + 8-hydroxyguanine (mutagenic)

Question 15

What is the most frequently mutated gene?

Answer 15

p53

Question 16

What does p53 do?

Answer 16

• crucial tumour suppressor gene (normally tied up with MDM2, which keeps p53 inactive)

When it is released from MDM2, it 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 the problem
• SEVERE stress, p53 can activate an apoptotic pathway by interacting with apoptosis proteins

Question 17

What are the types of DNA repair?

Answer 17

1. Direct reversal of DNA damage
   - Photolyase splits cyclobutane pyrimidine-dimers formed from UV light to recover the pyrimidines
   - Methyltransferases & alkyltransferases remove alkyl groups from DNA bases -> REAPIR
2. Base excision repair (mainly for apurinic/apyrimidinic damage)
   - DNA glycosylases & apurinic/apyrimidinic endonucleases and other enzyme partners affect repair
   - A repair polymerase (e.g. Polb) fills the gap formed and fixes it -> DNA ligase completes the repair
3. Nucleotide excision repair (mainly for bulky DNA adducts)
   - Xeroderma pigmentosum proteins (XP proteins) assemble at the damage.
   - A stretch of nucleotides either side of the damage are excised by endonuclease and helicase removes patch
   - Repair polymerases (e.g. Pold/b) fill the gap and DNA ligase completes the repair

*energy demandning, requires lots of proteins

4. During- or post-replication repair
   - Mismatch repair
   - Recombinational repair
   - These proteins check the DNA to make sure that it is ok before the daughter cells bud off in mitosis

Question 18

How long does the bacterial (ames) test for mutagenicity chemicals take?

Answer 18

matter of days

Question 19

How is the bacterial (ames) test for mutagenicity chemicals carries out?

Answer 19

Normally, Salmonella typhimurium is used for this assay

• incubate the chemical with rat liver enzymes (containing P450) to generate the reactive chemical
• these bacteria require exogenous histidine to be able to grow (genetically engineered so they can’t produce histidine)
• Using chemical mutagens, these cells can be mutated so that they regain the ability to produce histidine so cells can grow in the absence of histidine
• Mix bacteria with the activated chemical -> put them on a plate (which doesn’t have any histidine on it)
• Anything that has NOT been mutated will need exogenous histidine to grow and hence will die
• The mutated bacteria and regained the capacity to produce histidine will grow and survive
• The more the DNA damaging capability of the chemical, the more colonies grow in histidine absence
• This is a very quantitative assay, and also shows a good dose-response relationship

Question 20

Hoe can you detect chromosomal aberrations?

Answer 20

You incubate the cells with the DNA-damaging chemical. Then, you do karyotyping or you look at the chromosomal structure

You can look for chromatid exchange, chromatid gaps etc.

*process is slow, laborious and requires a lot of technical skill

Question 21

How is an in vitro micronucleus assay carrie dout?

Answer 21

We need the cell to go through one replication cycle and then stop it when a binucleus is formed.

*Cytochalasin-B is used to block cytokinesis and hold the cell in the binucleate stage.

Then the binucleate cells are assessed for the presence of micronuclei.

Micronuclei: pieces of chromosomal material that have been broken off and no longer appear in the nucleus. (normal = binucleus)

Question 22

What are the kenetochores of the chromosomes stained to determine?

Answer 22

e if chemical treatment caused:

• Clastogenicity (chromosomal breakage)
• Aneuploidy (chromosomal loss/change in the number of chromosomes)

Question 23

What happens in a bone marrow micronucleus assay in mice or rats?

Answer 23

• The animals are treated with the chemical
• Bone marrow cells or peripheral erythrocytes are examined for the presence of micronuclei
• The erythrocytes normally remove the nucleus during development
• But they CAN NOT remove small fragments of DNA (e.g. a micronucleus)

*If the chemical can generate small fragments of DNA as the erythrocytes are formed from the pluripotent stem cells, these fragments will persist. The presence of micronuclei in the erythrocytes indicates DNA damage.