DNA damage and repair

Question 1

What can damage DNA?

Answer 1

Chemicals - dietary (40%), medical, lifestyle, environmental, occupational, endogenous
Radiation - ionising, solar, cosmic

Question 2

What kind of damage can occur to DNA?

Answer 2

> base dimers and chemical cross links
DNA adducts and alkylation)
base hydroxylation and abasic sites
double/single strand breaks

Question 3

What is deamination?

Answer 3

primary amino groups converted to ketogroups

Question 4

What are chemical modifications?

Answer 4

Several types of hyper reactive oxygen species can modify DNA bases

Question 5

What is photodamage

Answer 5

UV light absorbed by nuclei acid bases causes influx of energy which can induce chemical changes. - most commonly bond formation between adjacent pyrimidines within one strand

Question 6

Where do polycyclic aromatic hydrocarbons (Benzo[a]pyrenes/B[a]Ps) originate from?

Answer 6

Common environmental pollutants
Formed from combustion of fossil fuels
Formed from combustion of tobacco

Question 7

What happens in the first phase of mammalian metabolism and where does it occur

Answer 7

>  Phase 1 – addition of functional groups – oxidations, reductions, hydrolysis.
o Mediated mainly by cytochrome p450 enzymes.

Question 8

What happens in the second phase of mammalian metabolism and where does it occur

Answer 8

 Phase 2 – conjugation of phase 1 functional groups – glucuronidation, sulphation, glutathione conjugation, methylation, acetylation & amino-acid conjugation.
o Generates polar (water soluble) metabolites to excrete.

Question 9

How is B[a]P epoxidated?

Answer 9

 B[a]P epoxidation process:
o P450 enzymes oxidise the B[a]P (becomes very reactive).
o EH (epoxide hydrolase) removes the toxic oxide.
o P450 again oxidises the B[a]P which then degrades spontaneously.
o +ve-charged B[a]P then adducts onto DNA.

Question 10

How is aflatoxin B1 epoxidated?

Answer 10

 Aflatoxin B1 epoxidation process:
o P450 oxidises the aflatoxin B1.
o Aflatoxin B1 the adducts to DNA directly using its adjacent N7 positively charged carbon atom.

Question 11

What are some sources of aflatoxin B1

Answer 11

o Formed by Aspergillus flavus mould and is commonly found in poorly stored grains and peanuts.
o Is a potent liver carcinogen (in Africa/far-east).

Question 12

What is 2-naphthylamine and where does it come from?

Answer 12

 2-naphthylamine:
o A past component of dye-stuffs and includes benzidine.
o 2-nap is a potent human bladder carcinogen.

Question 13

How is 2-naphthylamine metabolised and cause DNA damage?

Answer 13

 2-nap metabolism:
o Cytochrome P1A2 oxidises the amine group.
o Glucuronyl transferase adds a glucuronide group to the amine which is the broken by the acidic urine pH.
o The nitrenium ion remaining then causes DNA damage in the bladder.

Question 14

How does solar (UV) radiation cause DNA damage?

Answer 14

o UV radiation stimulates formation of pyrimidine (thymine) dimerisation  skin cancer.

Question 15

How does ionising radiation cause DNA damage>?

Answer 15

 Ionising radiation:
o Generates free radicals inside cells such as oxygen free radicals – super oxide (O2•) and hydroxyl (HO•).
o These oxygen free radicals have unpaired electrons that are electrophilic and so seek out electron-rich DNA (negatively charged sugar-phosphate backbone of DNA).

Question 16

What kind of damage do free radicals do to DNA>

Answer 16

o DNA damage from oxygen free radicals includes:
 Double/single stand breaks.
 Apurinic & apyrimidinic sites – sites where the base is lost whilst the backbone remains.
 Base modifications:
• Ring-opening – guanine & adenine.
• Glycol (unstable products of oxidation) formation – thymine & cytosine.
• Creation of 8-hydroxyadenine & 8-hydroxyguanine – mutagenic.

Question 17

How does p53 deal with cellular stress?

Answer 17

p53 can sense the DNA damage and regulate the response of the cell to this event.

Question 18

Describe direct reversal of DNA repair

Answer 18

o Photolyase splits cyclobutane pyrimidine dimers.

o Methyltransferases & alkyltransferases remove alkyl groups from the bases.

Question 19

 Base excision repair – mainly for apurinic & apyrimidinic damage:

Answer 19

o DNA glycosylases & apurinic/apyrimidinic endonucleases.
o A repair polymerase (e.g. Pol-beta) fills the gap and DNA ligase completes the repair.

Question 20

 Nucleotide excision repair – mainly for bulky DNA adducts:

Answer 20

o Xeroderma pigmentosum proteins (XP proteins) assemble at damage  stretch of nucleotides are excised.
o A repair polymerase (e.g. Pol-beta) fills the gap and DNA ligase completes the repair.

Question 21

 During- or post-replication repair:

Answer 21

o Mismatch repair. - scrutinisation of DNA for a-posed bases that do not pair properly
o Re-combinational repair.

Question 22

What is the pathway in base excision repair?

Answer 22

o DNA-glycosylase – removes the base.
o AP-endonuclease – cuts the backbone.
o DNA polymerase – adds a complimentary base.
o DNA ligase – re-joins the backbone.

Question 23

What is the pathway in nucleotide excision repair?

Answer 23

o Endonuclease – cuts a large swathe of backbone.
o Helicase – removes 1+ nucleotides.
o DNA polymerase – adds complimentary bases.
o DNA ligase – re-joins the backbone.

Question 24

What is the pathway in nucleotide excision repair?

Answer 24

o Endonuclease – cuts a large swathe of backbone.
o Helicase – removes 1+ nucleotides.
o DNA polymerase – adds complimentary bases.
o DNA ligase – re-joins the backbone.

Question 25

What are some human generic diseases involving NER? (MODIFY)

Answer 25

Xeroderma pigmentosum

Question 26

When are DNA double strand breaks made?

Answer 26

Under physiological conditions during somatic recombination and transposition: eg: VDJ recombination
During homologous recombination
As a result of ionising radiation and oxidative stress induced DNA damage

Question 27

What are the fates of carcinogen damage leading to altered DNA

Answer 27

1. Efficient repair - normal cell
2. Apoptosis
3. Incorrect repair -> DNA replication & cell division (fixed mutation) -> either:
   a. Transcription/translation to aberrant proteins.
   b. Carcinogenesis if critical targets are mutated (e.g. proto-oncogenes and TSGs).

Question 28

How are new drugs tested for their effect on DNA? (PHOTO FROM SLIDE NEEDS TO BE ADDED)

Answer 28

When new drugs are made, they need to be tested to check for their effect on the DNA. The order of testing is shown to the left.

1. SAR – checking the molecule structurally for groups that could precipitate cancer.
2. Bacterial gene mutation assay (IN VITRO).
3. Mammalian cell assay (IN VITRO).
4. Mammalian assay (IN VIVO).
5. Investigate mammalian assays.

Question 29

How is the bacterial (Ames) test carried out and why? - (RESEARCH PLS!)

Answer 29

 The rat liver enzymes are used to activate (metabolise) the potential carcinogen so that it becomes potentially toxic.
 The bacteria are modified so that they do not produce histidine and so require exogenous histidine to grow and survive.
 If the bacteria mutate with the chemical, they can regain the ability to produce histidine and so will grow even without exogenous histidine.

Question 30

How is DNA damage tested for in mammalian cells?

Answer 30

 Mammalian cells with the chemical in the presence of liver S9 enzymes are inspected directly for chromosomal damage.

Question 31

How is DNA damage tested for in micronucleus assays?

Answer 31

 Mammalian cells are treated with the chemical and allowed to divide.
 Cytokinesis is blocked using cytochalasin-B.
 Bi-nucleate cells are assessed for the presence of micronuclei.
 The kinetochore proteins are stained to determine if the chemical treatment caused:
o Clastgenicity – chromosomal breakage.
o Aneuploidy – chromosomal loss/gain.

Question 32

How is DNA damage tested for in bone marrow micronucleus tests?

Answer 32

 Animals are treated with the chemical and the bone marrow cells or peripheral blood cells are examined for micronuclei.
 The erythrocytes can usually remove the nucleus during development but CANNOT remove the small fragments of DNA (of which the cell forms a micronucleus around).