DNA Damage and Repair

Question 1

How can DNA become damaged by endogenous (internal) sources?

Answer 1

1. Replicative errors:
   - point mutation
   - insertion
   - deletions
2. Oxidative damage by free radicals (oxygen metabolism)
3. Spontaneous alteration in DNA:
   - base loss
   - deamination (change in the base)
4. Alkylating agents:
   - malondialdehyde (byproduct of polyunsaturated fatty acid peroxidation and arachidonic acid

Question 2

How can DNA become damaged by exogenous (external) sources?

Answer 2

1. UV -> intrastrand cross links
2. Pollution:
   - hydrocarbons -> base modification
3. Carcinogens -> base modification
4. Radiotherapy:
   - ionising/X-rays -> ss and ds DNA breaks
5. Chemotherapy:
   - alkylating agents -> base modification
   - cisplatin -> interstrand cross links
   - mitomycin X -> DNA cross links/base modifications
   - cyclophosphamide -> base modification
   - psoralen -> interstrand cross links
   - melphalan -> DNA cross links/base modifications

Question 3

What sort of changes to DNA occur?

Answer 3

1. Intrastrand crosslink -> 2 bases on the same strand become covalently attached
2. Ss/ds break -> lose one or both strands and all the info they contain
3. Base change -> eg deamination
4. Base loss -> eg depurination
5. Interstrand crosslink -> 2 bases on opposite strands are covalently attached so they can’t separate and can’t be transcribed
6. Base modification -> adding a random chemical base (eg O6, CH3)
7. Pyrimidine dimer -> specific intrastrand crosslink of 2 adjacent pyrimidines

Question 4

What processes can help with DNA damage errors?

Answer 4

1. Direct reversals -> can reverse the action (eg pyrimidine dimer)
2. Nucleotide excision repair (NER) -> error is remove as a stretch of nucleotides using enzymes
3. Base excision repair (BER) -> only the affected base is removed using enzymes
4. Mismatch repair (MMR) -> corrects nucleotides that are mismatched by removing the incorrect one from the new strand using Mut enzymes

Question 5

Which homologous recombination processes help specifically with strand invasion?

Answer 5

1. Double strand breaks (DSBR)-> only occurs in S phase when homologous chromosomes are present so occurs in late S/G2
2. Synthesis-dependent strand-annealing (SDSA) -> occurs during mitosis late in S/G2 and replaces the sequence around a DNA double-strand break (DSB) with a copy of a homologous DNA template
3. Single-strand annealing (SSA) -> only occurs when there are adjacent repeats and repairs double-strand breaks between two repeat sequences
4. Break induced replication (BIR) -> only occurs when there is 1 end with homology eg during DNA replication ahead of the fork or outside S phase
5. Microhomology mediated end joining (MMEJ) -> occurs throughout S phase and uses short homologous sequences (microhomologies) to align the broken ends prior to ligation
6. Non homologous end joining -> occurs early S phase before replication and break ends are directly ligated without the need for a homologous template

Question 6

Which processes can help with DNA replication errors?

Answer 6

1. Translesion synthesis (TLS) -> process by which cells copy DNA containing unrepaired damage that blocks progression of the replication fork
2. Interstrand crosslinks (ICL) repair -> crosslinks between sister strands

Question 7

What functions allow the BRCA1 gene to have genome stability?

Answer 7

1. DNA damage repair
2. Transcriptional regulation
3. Cell cycle checkpoint
4. Protein ubiquitination

Question 8

What happens if repair pathways fail?

Answer 8

1. Point mutations (altered gene expression)
2. Ss or ds DNA breaks (interfer with replication/rearrange chromosomes)
3. Failure to stimulate senescence
4. Failure to stimulate apoptosis

Question 9

List examples of repair genes in cancer

Answer 9

1. p53
2. BRCA1/2 -> breast/ovarian cancer
3. ATM -> leukaemia, lymphoma
4. BLM -> leukaemia, lymphoma
5. XPC/XPE -> skin cancer

Question 10

Why are there so many different mechanisms for DNA damage repair?

Answer 10

• Mechanisms are only employed when there are problems in the cell
• Don’t necessarily occur in every cell cycle
• Therefore the cell is more likely to be able to tolerate these chase without lethality to the organism

Question 11

What are some repair proteins in the treatment of cancer?

Answer 11

1. Prognostic indicators -> stats of several repair genes correlated to poor prognosis
2. Drug targets -> if cells can’t repair their DNA they are usually driven to apoptosis which is ideal in some cases eg BRCA2 cells with PARP inhibitors
3. Combination therapy -> targeting repair proteins sensitises cells for other forms of treatment which allows better targeting and lower doses of radiation/chemo