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

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.

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

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

Answer 11

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 12

What does UV radiation lead to the formation of?

Answer 12

Pyrimidine (thymine) dimers – adjacent pyrimidines can covalently link

Question 13

What does ionising radiation generate?

Answer 13

Free radicals

Question 14

Name 2 oxygen free radicals.

Answer 14

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

Question 15

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

Answer 15

Single and Double strand breaks
Apurinic and apyrimidic sites
Base modifications

Question 16

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

Answer 16

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

Question 17

What are the main types of DNA repair?

Answer 17

Direct reversal of DNA damage
Base excision repair
Nucleotide excision repair
During- and post-replication repair

Question 18

Give two examples of direct reversal of DNA damage.

Answer 18

Photolyase looks for cyclobutane-pyrimidine dimers and cuts them
Methyltransferases and alkyltransferases remove alkyl groups from the bases

Question 19

What comes under during and post replication repair?

Answer 19

Mismatch repair

Recombinational repair

Question 20

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

Answer 20

Guanine

Question 21

Describe the process of base excision repair.

Answer 21

DNA glycosylase hydrolyses between the base and the sugar
Then AP endonuclease splits the DNA strand so there is a gap in the backbone
DNA polymerase then fills in the missing base (using the complementary strand as template)
DNA ligase then seals the DNA

Question 22

Describe the process of nucleotide excision repair.

Answer 22

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, leaving the double strand with a patch missing
DNA polymerase replaces the missing bases
DNA ligase joins the DNA up

Question 23

Describe the possible fates of carcinogen-DNA damage.

Answer 23

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) –> transcription and translation giving aberrant proteins + carcinogenesis if critical targets are mutated

Question 24

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

Answer 24

Look at structure of compound
Test in vitro on bacteria
Test in vitro on mammalian cells- stem cells used
Test in vivo on mammals

Question 25

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

Answer 25

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 26

Describe the use of in vitro micronucleus assays.

Answer 26

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 27

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

Answer 27

Clastogenicity – chromosomal breakage

Aneuploidy – chromosomal loss/change in the number of chromosomes

Question 28

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

Answer 28

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 29

What happens to p53 production under no stress

Answer 29

MDM2 suppresses its transcription and any that is produced gets degraded by proteasomes. p53 also activates transcription of MDM2 leading to a negative feedback loop

Question 30

What happens to p53 production under stress

Answer 30

MDM2 gets phosphorylated (inactivated) leading to an increase in levels of p53

Question 31

What stresses cause an increase in p53

Answer 31

Hypoxia
Radiation
DNA adducts

Question 32

What does p53 do when cell is under stress

Answer 32

Activates pathways either leadig to DNA repair or apoptosis if damage too bad

Question 33

Where does Alfatoxin B1 come from

Answer 33

Aspergillus flavus mould- normally from poorly stored grain and peanuts from Asia and Africa

Question 34

Where is Alfatoxin B1 most toxic

Answer 34

Liver

Question 35

1st stage in metabolism of Alfatoxin B1

Answer 35

C=C is attacked by CP450 to form Alfatoxin B1,2,3-epoxide

Question 36

Problem of alfatoxin B1,2,3-epoxide

Answer 36

Not a good substrate for epoxide hydrolase so persists for a while in liver and attacks guanine residues