DNA damage

Question 1

How common is DNA damage events and how can it lead to tumour formation?

Answer 1

• • Human genome consists of 3x10^9 base pairs and DNA damage occurs at a rate of 10^3-10^6 molecular lesions per cell per day.
• • Can either repair DNA damage or eliminate damaged cells – this depends on how important the cell is (germ cells and stem cells) and how beneficial it would be.
• • Cells are continuously exposed to DNA damaging events but due to the efficiency of repair mechanisms, only a few will become somatically acquired mutations.
• • For the development of cancer, mutations in the critical genes – oncogenes (gain of function mutations) and tumour suppressor genes (loss of function mutations) are required.
• • It takes a very long time for a set of critical gene mutations to occur in a single cell which is why cancer is mainly a disease of old age.
• • Mutations in DNA repair enzymes will promote genomic instability and can lead to cancer.

Question 2

What are the eight types of DNA damage?

Answer 2

1. Deamination – removal of an amine group from a molecule. For example cytosine is deaminated to uracil. Deamination can lead to mismatch errors in the DNA strand.
2. Tautomerisation: spontaneous isomerisation involving migration of a hydrogen atom/proton. e.g. guanine’s keto form to guanine’s enol form. It can also lead to mismatch pairing.
3. Oxidation: damaging bases by free radicals of oxygen
4. Alkylation: addition of alkyl groups to bases or backbone DNA (e.g. O6-me). Can be caused by a number of chemotherapy agents.
5. Depurination: loss of Adenine or Guanine bases: 10 000 bases per day per cell
6. Pyrimidine dimers: by sunlight (UV light) – covalent linkage between two pyrimidine bases. Can be caused by excessive tanning.
7. Single or double strand break: by ionising radiation. Double strand break is much harder to repair.
8. DNA replication errors: lack of fidelity

Question 3

What are the two steps of chemical carcinogenesis?

Answer 3

1. Initiation - induction of mutations in cell which under certain conditions allow them to be later transformed. Not sufficient to transform a cell.
   - – Targets of initiation include proto-oncogenes, TSGs and apoptosis regulating genes.
   - – They can be direct acting carcinogens – which don’t need activation or procarcinogens which require metabolic activation by the host to become active, e.g. by cytochrome P-450 oxygenases.
   - – The mutation needs to occur in a dividing cell in order to form tumour.
2. Promotion - further induction of proliferation of initiated cell, often involves a number of normalgrowth signals.
   - – Usually the initiated cell is primed to be more sensitive to these signals – causing uncontrollable proliferation, leading to a lot more cells and more mutations. Formation of tumour occurs.

Question 4

What are examples of chemical carcinogens?

Answer 4

1. Alkylating agents – they alkylate genetic material resulting in cell death; they are used in chemotherapy and immune suppression; shown to increase the risk of tumors later on, particularly lymphoid & leukemic neoplasms.
2. Polycyclic aromatic hydrocarbons: particularly potent carcinogens found in cigarette smoke.
   - –> Benzo(a)pyrene is an example of one, its metabolite forms an adduct linked to lung cancer mutations.
3. Naturally occurring carcinogens: Aflatoxin B1 produced by Aspergillus flavus fungi (favors grains & peanuts) is a potent hepatocarcinogen associated with liver cancer in China & Africa.
4. Nitrosoamines & amides: Ingested as nitrites or derived from digested proteins may contribute to gastric cancer. Found in meats, tobacco smoke. Nitrites are used to cure meats (hot dogs, pastrami, etc.) they are also produced from proteins exposed to high temperatures – frying.

Question 5

DNA repair? What are the types of DNA damage reversal?

Answer 5

1. Photo-reactivation: pyrimidine dimers. Pyrimidine dimers distort the DNA structure and prevent transcription/replication. Photolyase enzyme will split the dimers in presence of light.
2. Damage base repair: involves repairing mismatched bases by enzymes without breaking the DNA backbone.
   - —> Deamination – Uracil N-glycosylase removes uracil formed by cytosine deamination.
   - —> Alkylation – repaired by transfer of methyl group by Methyl Guanine-DNA MethylTransferase (MGMT), and we will have Guanine. MGMT is a suicide enzyme, once it removes one alkyl group it can no longer catalyse and it’s removed from the cell.
3. Ligation of simple strand breaks: are repaired by DNA ligase.

Question 6

List the types of DNA damage removal.

Answer 6

1. Base excision repair
2. Nucleotide excision repair
3. Mismatch repair

Question 7

Describe the mechanism of base excision repair.

Answer 7

• • It’s the predominant mechanism that deals with spontaneous DNA damage caused by free radicals.
• • It repairs small, non-bulky (i.e. no distortion to the helix) DNA lesions (includes methylated, oxidized, reduced bases).

Mechanism:-

1. Recognition of the damaged base by a DNA glycosylase. Eight enzymes –> each one responsible for identifying and removing a specific kind of base damage.
2. Removal by cleaving the covalent bond between base + backbone, producing a gap: an AP site - apurinic/apyrimidinic site, also known as an abasic site, is a location in DNA that has neither a purine nor a pyrimidine base, either spontaneously or due to DNA damage
3. Resynthesis with the correct nucleotide. Done by DNA polymerase beta using the other strand as a template.
4. Religation of the break in the strand.

Question 8

Describe the mechanism of nucleotide excision repair.

Answer 8

• • It is a more complicated process than BER.
• • Mediated by 30 distinct proteins that function as a large enzyme complex called the nucleotide excision repairosome.
• • The sole repair system for bulky DNA lesions (e.g. crosslinks in DNA), which creates a block to DNA replication and transcription.

Mechanism:-

1. Recognition of damage by one or more protein factors and assembly of nucleotide excision repairosome.
2. Removal by double incision of the damaged strand by an endonuclease and removal of the short segment (~20 bases) containing the damaged region by an exonuclease.
3. Resynthesis of the resulting gap by DNA polymerase beta: synthesizes DNA using the opposite strand as a template.
4. Religation: a DNA ligase binds the synthesized piece into the backbone.

Question 9

Describe the mechanism of mismatch repair.

Answer 9

Repairs the mismatches that are a result of DNA proofreading failure.
– Mutations in the several of these genes are associated with hereditary non-polyposis colon cancer.

Mechanism:-

1. Recognition by group of repair proteins which can scan DNA and look for incorrectly paired bases.
2. Removal by an exonuclease – removes a patch of DNA.
3. Resynthesis of the repair patch is done by a DNA polymerase using other as template.
4. Religation of remaining single strand break

Question 10

Why are double strand breaks so important to repair?

Answer 10

• • DSBs are a result of ionising radiation and ROS and are highly deleterious – can lead to chromosomal rearrangements.
• • DSB’s are a major cytotoxic lesion: even a single unrepaired DSB can lead to cell death.

Question 11

What are the two mechanisms of DSB repair?

Answer 11

• -DSBs are repaired by two different mechanisms:
  1. End joining repair: DNA ligase IV joins broken chromosome ends in a way that is not dependent on sequence homology, therefore is not error free. Incorrect ends can join and this causes introduction of mutations —> non-homologous end joining.

2. Recombination repair: preferred mechanism of repair since it is least likely to result in mutations: broken ends are repaired using the information on the intact homologous chromosome. Dependent on sequence homology and therefore error free.

Question 12

Give some examples of disorders that are related to defects in DNA repair.

Answer 12

1. Xeroderma pigmentosum – caused by defects in the NER system. It’s a rare genetic skin – the skin is abnormally sensitive to UV light. It is caused by DNA damage – there’s a lack in NER of UV-induced thymine dimers, i.e. an endonuclease deficiency. Skin tumours are formed.
2. There are a lot more disorders associated with defects in DNA damage and are associated with predisposition to cancer, e.g. ataxia telangiectasia.
3. There’s also defective double strand break repair in familial breast cancer genes  BRCA1 and BRCA2.

Question 13

Explain the mechanism of PARP inhibitors in relation to the BRCA genes.

Answer 13

They are drugs that are pharmalogical inhibitors of the enzyme poly ADP ribose polymerase (PARP).

• • PARP is important for repairing single-strand breaks (‘nicks’ in the DNA).
• • PARP inhibitors cause the nick to persist unrepaired until DNA is replicated (which must precede cell division), then the replication itself can cause double strand breaks to form.
• • BRCA1, BRCA2 are proteins that are important for the repair of double-strand DNA breaks by homologous recombination. If mutated, the change can lead to errors in DNA repair that can eventually cause breast cancer.
• • Drugs that inhibit PARP1 can cause multiple double strand breaks to form in this way, and in tumours with BRCA1, BRCA2 mutations these double strand breaks cannot be efficiently repaired, leading to the death of the cells.
• • This is an effective way of targeting breast cancer cells.
• • Normal cells that don’t replicate their DNA as often as cancer cells, and that lacks any mutated BRCA1 or BRCA2 still have homologous repair operating, which allows them to survive the inhibition of PARP.