DNA Damage and Repair

Question 1

State some different types of DNA damage caused by carcinogens.

Answer 1

Base dimers and chemical cross-links

Base hydroxylations

Abasic sites

Single strand breaks

Double strand breaks

DNA adducts & alkylation- addition of large carcinogenic groups

Question 2

What are abasic sites?

Answer 2

During the repair process, the entire DNA base has been removed so the sugar backbone is maintained but we have removed the base from the mutagenic molecule

During replication, this missing base can cause problems

Question 3

What are the implications of single strand breaks?

Answer 3

These are common and useful

Topoisomerase causes single strand breaks and it is involved in relaxing and unwinding the DNA before replication

Question 4

What are the implications of double strand breaks?

Answer 4

These are NOT GOOD

The two strands have a tendency to drift apart when a double strand break occurs

There are repair mechanisms for dealing with this, but sometimes the DNA repair can go wrong and introduce DNA damage

Question 5

What is the usual type of damage that is caused by chemicals?

Answer 5

DNA adducts

Question 6

Why is DNA the target for many carcinogens?

Answer 6

Chemical carcinogens are usually metabolically activated and converted into electrophiles (they want electrons)

DNA is very electron rich

Question 7

What are the consequences of bulky DNA adducts?

Answer 7

The electrophiles bind and form a covalent bond

The binding of these adducts causes problems, particularly during replication because it interferes with the ability of DNA polymerase to recognise the base (because of the bulky adduct)

Question 8

What are the six types of Phase II reaction?

Answer 8

Glucuronidation,   
Acetylation,   
Sulphation,  
Methylation,   
Amino acid conjugation,   
Glutathione conjugation

Question 9

What are polycyclic aromatic hydrocarbons?

Answer 9

They are environmental pollutants formed from the combustion of fossil fuels and tobacco

Question 10

Describe the two-step oxidation of benzo[a]pyrene (B[a]P).

Answer 10

B[a]P is a substrate for CYP450, which converts it to B[a]P-7,8-oxide (this is an electrophile)

The body has a defence mechanism – epoxide hydrolase converts the oxide to a dihydrodiol (B[a]P-7,8-dihydrodiol)

This is inactive

However, this dihydrodiol is also a substrate for CYP450, which converts it to another oxide (B[a]P-7,8-dihydrodiol-9,10-oxide)

This even more reactive than the previous oxide – it goes on to form DNA adducts

Question 11

State two past components of dyestuff that are potent bladder carcinogens.

Answer 11

Benzidine and 2-naphthylamine

Question 12

Explain the mechanism by which 2-naphthylamine is a bladder carcinogen.

Answer 12

2-naphthylamine is converted by CYP450 to a hydroxylamine derivative, which is reactive

In the liver, this is glucuronidated (thus inactivating it)

The inactive metabolite is excreted by the liver and then it goes to the bladder where it mixes with the urine

The ACIDITY of the urine causes hydrolysis of the glucuronides – this releases the hydroxylamine derivative, which forms a nitrenium ion

This is electrophilic so it leads to the formation of DNA adducts

Question 13

What does UV radiation lead to the formation of?

Answer 13

Pyrimidine (thymine) dimers – adjacent pyrimidines can covalently link

Question 14

What does ionising radiation generate?

Answer 14

Free radicals (Unpaired electrons are electrophilic so seek electron rich DNA—>DNA damage)

Question 15

Name 2 oxygen free radicals.

Answer 15

Superoxide radical (O2.) 
Hydroxyl radical (HO.)

Question 16

What are the consequences of oxygen free radical attack on DNA?

Answer 16

Single and Double strand breaks

Apurinic and apyrimidic- sites with no bases

Base modifications:
 Ring-opened guanine and adenine
 Thymine and cytosine glycols
 8-hydroxyadenine and 8-hydroxyguanine

Question 17

What are the p53 mediated responses to mild and severe physiological stress?

Answer 17

Mild – repair the damage and restore the normal function of the cell

Severe – apoptosis

Question 18

What are the main types of DNA repair?

Answer 18

Direct reversal of DNA damage

Base excision repair

Nucleotide excision repair

During- and post-replication repair

Question 19

Give two examples of direct reversal of DNA damage.

Answer 19

Photolyase looks for cyclobutane-pyrimidine dimers and cuts them

Methyltransferases and alkyltransferases remove alkyl groups from the bases

Question 20

What comes under during and post replication repair?

Answer 20

Mismatch repair

Recombinational repair

Question 21

Which base is most electron-rich and hence most capable of attracting electrophiles?

Answer 21

Guanine

Question 22

Describe the process of base excision repair.

Answer 22

DNA glycosylase hydrolyses between the base and the sugar- removes base

Then AP endonuclease splits the DNA strand so there is a gap in the backbone (cuts backbone)

DNA polymerase then fills in the missing base (using the complementary strand as template) - adds complimentary base

DNA ligase then seals the DNA- rejoins backbone

Question 23

Describe the process of nucleotide excision repair.

Answer 23

Xeroderma pigmentosum proteins (XP proteins) assemble at damage site

Endonuclease makes two cuts in the DNA on either side of the site of damage (this demarcates a patch of DNA)-

Helicase then removes this patch (multiple nucleotides), leaving the double strand with a patch missing

DNA polymerase replaces the missing nucleotides (bases + backbone)

DNA ligase joins the DNA up - ie connects the backbone

Question 24

Describe the possible fates of carcinogen-DNA damage.

Answer 24

Low level of damage –> effective repair –> return to being a normal cell

Severe damage –> apoptosis

Carcinogen causing altered DNA –> incorrect repair/altered primary sequence –> DNA replication and cell division (fixed mutation) –> :
o transcription and translation giving aberrant proteins or o carcinogenesis if critical targets are mutated

Question 25

Describe the process of testing whether a chemical can cause carcinogenesis.

Answer 25

Look at structure of compound (SAR- structure activity relationship)

Test in vitro on bacteria

Test in vitro on mammalian cells

Test in vivo on mammals

investigate in vivo mammalian assays

Question 26

Describe the bacterial (Ames) test for mutagenicity of chemicals.

Answer 26

This test usually uses Salmonella typhimurium
The bacterium is genetically engineered so that it can’t produce histidine, so it can only survive and grow on a culture medium that has exogenous histidine

The compound to be tested is, firstly, incubated with rat liver enzymes containing CYP450 enzymes to metabolise the chemical into an active form that can be carcinogenic

The bacteria are mixed with the active chemical and then placed on a culture medium with NO histidine

Any colonies that survive will have become mutated by the chemical so that it regains the ability to produce its own histidine and hence cangrow in the absence of histidine

Any bacteria that hasn’t been mutated will die on the dish
The greater the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine

Question 27

Describe the use of in vitro micronucleus assays.

Answer 27

This is trying to measure the ability of a chemical to break up DNA into fragments

We need the cell to go through one replication cycle and then stop it when it’s at the binucleus stage – this is when you check for the presence of micronuclei

Question 28

What is used to block cytokinesis and hold the cell in the binucleate stage in the micronucleus assay?

Answer 28

Cytochalasin-B

Question 29

What are the two types of chromosomal damage that can be detected by this assay?

Answer 29

Clastogenicity – chromosomal breakage

Aneuploidy – chromosomal loss/change in the number of chromosomes

Question 30

Explain the reasoning behind the use of bone marrow micronucleus assays to test the mutagenicity of a chemical.

Answer 30

Bone marrow is pluripotent

The animals are treated with the chemical and their bone marrow cells and peripheral erythrocytes are examined for the presence of micronuclei

Erythrocytes normally remove the nucleus during development, but it CANNOT remove small fragments of DNA e.g. a micronucleus

So the presence of micronuclei in erythrocytes indicates DNA damage

Question 31

what can damage DNA

Answer 31

Chemicals

Radiation

Question 32

give an example of a potent liver carcinogen

Answer 32

Alfatoxin B1- produced by aspergillus flavus mould and is found on poorly stored grains and peanuts mould (mostly in africa)

Question 33

describe the Aflatoxin B1 epoxidation process

Answer 33

P450 oxidises the aflatoxin b1

aflatoxin then adducts the DNA directly using its adjacent N7 positively charged carbon atom

Question 34

what does base excision repair fix

Answer 34

mainly apurinic and apyriminidinic damage

Question 35

what does nucleotide excision repair fix

Answer 35

mainly bulky DNA adducts