DNA Damage & Repair

Question 1

What can damage DNA?

Answer 1

Chemicals
• dietary (40%)
• medical
• lifestyle

Radiation
• ionising
• solar
• cosmic

Question 2

Types of DNA damage?

Answer 2

DNA double helix
Deamination
Chemical modification
Photodamage

Question 3

Why is DNA damage so important?

Answer 3

Can lead to mutations
• which can lead to cancer

HENCE
Damaging DNA is an important strategy in cancer therapy

Question 4

How can carcinogens damage DNA - the 4 mechanisms?

Answer 4

DNA adducts & alkylation
• addition of large carcinogenic groups

Base dimers & chemical cross-links

Base hydroxylation and abasic (base removed) sites form

Double/single strand breaks

Question 5

Briefly explain mammalian metabolism

Answer 5

2 phases!

Phase 1 - addition of functional groups
• oxidation, reductions, hydrolysis
• mediated mainly by cytochrome p450 enzymes

Phase 2 - conjugations of Phase 1 functional groups
• sulphation, glucuronidation, acetylation, mehtylation
• generates POLAR (water soluble) metabolites to excrete

Question 6

Fact about carcinogens and metabolism?

Answer 6

Most carcinogens are insidious AND only become carcinogenic AFTER phase 1 metabolism

Question 7

Explain the metabolism of Polycyclic Aromatic Hydrocarbons

Answer 7

Polycyclic Aromatic Hydrocarbons – common environmental pollutants formed from combustion of fossil fuels or tobacco

2-step B[a]P EPOXIDATION process:
• P450 enzymes oxidise the B[a]P (becomes very reactive)
• EH (epoxide hydrolase) removes the toxic oxide
• P450 again oxidises the B[a]P which then degrades spontaneously
• +VE-charged B[a]P then adducts onto DNA

B[a]P = Benzo[a]pyrene

Question 8

Explain the metabolism of Aflatoxin B1

Answer 8

EPOXIDATION

Aflatoxin B1:
• formed by Aspergillus flavus mould and is commonly found in poorly stored grains and peanuts
• is a potent liver carcinogen (in Africa/far-east)

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

Question 9

Explain the metabolism of 2-naphthylamine

Answer 9

2-naphthylamine:
• a past component of dye-stuffs and includes benzidine
• 2-nap is a potent human bladder carcinogen

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

Question 10

Explain the metabolism of other carcinogens - solar (UV) radiation?

Answer 10

UV radiation stimulates formation of pyrimidine (thymine) dimers
• causes skin cancer

Question 11

Explain the metabolism of other carcinogens - ionising radiation

Answer 11

Generates free radicals inside cells such as oxygen free radicals
• super oxide (O2•)
• hydroxyl (HO•)

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 12

How can oxygen free radicals attack/damage DNA?

Answer 12

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 13

Relationship between DNA damage and mutation?

Answer 13

The greater the persistence of damage then the greater the chance of a mutagenic event

Question 14

What enzyme is key for DNA damage repair?

Answer 14

p53

Can sense the DNA damage and regulate the response of the cell to this event

(onenote picture!!)

Question 15

4 broad types of DNA repair?

Answer 15

Direct reversal of DNA damage – e.g.:

• Photolyase splits cyclobutane pyrimidine dimers (repair thymine dimers)
• Methyltransferases & alkyltransferases remove alkyl groups from the bases (reverse simple alkylation adducts)

Base excision repair – mainly for apurinic & apyrimidinic damage:

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

Nucleotide excision repair – mainly for bulky DNA adducts:

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

During- or post-replication repair:

• Mismatch repair - compare old and new strand (prefer new strand) and correct it
• Re-combinational repair.

Question 16

Two type of Exicision repair and enzymes that make up each pathways and are used to repair?

Answer 16

Base excision repair:
 • DNA-glycosylase – removes the base
 • AP-endonuclease – cuts the backbone
 • DNA polymerase – adds a complimentary base
 • DNA ligase – re-joins the backbone

Nucleotide excision repair:
• Endonuclease – cuts a large swathe of backbone
• Helicase – removes 1+ nucleotides
• DNA polymerase – adds complimentary bases
• DNA ligase – re-joins the backbone

Question 17

What are the most common damage to DNA that can be seen?

Answer 17

De-purination

&

Single-strand breaks

Question 18

Fate of Carcinogen-DNA damage (leading to altered DNS)?

Answer 18

Carcinogen damage leading to altered DNA can:
1. Repair

2. Apoptosis – If the damage is too much
3. Incorrect repair –> DNA replication & cell division (fixed mutation) –> :
   a. Transcription/translation to aberrant proteins.
   b. Carcinogenesis if critical targets are mutated (e.g. proto-oncogenes and TSGs).

Question 19

When new drugs are made, they need to be tested to check for their effects on the DNA - what is the order of testing that is carried out?

Answer 19

1. SAR
   • checking the molecule structurally for groups that could precipitate cancer
2. Bacterial gene mutation assay (IN VITRO)
   • e.g. Ames test w. S.typhimurium
3. Mammalian cell assay (IN VITRO)
   • e.g. chr. aberration
4. Mammalian assay (IN VIVO)
5. Investigate mammalian assays

Question 20

Explain how Bacterial gene mutation assay isused to test for DNA damage

Answer 20

IN VITRO - Ames

The rat liver enzymes (s9) 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 21

Explain how you can detect DNA damage in mammalian cells

Answer 21

Treat mammalian cells with the chemical in the presence of liver s9 enzymes
• inspected directly for chromosomal damage

Question 22

Explain how you can use Micronucleus Assays to test for DNA damage

Answer 22

IN VITRO

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:
• Clastgenicity – chromosomal breakage.
• Aneuploidy – chromosomal loss/gain.

Question 23

Explain how Bone Marrow Micronucleus tests can be used to test for DNA damage

Answer 23

IN VIVO

Animals are treated with the chemical & 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).