DNA Repair

Question 1

What is the DNA damage response (DDR)?

Answer 1

A signalling cascade in which:

1. SENSORS detect replication stress & DNA damage
2. TRANSDUCERS (= central DDR kinases) pass on the signal to
3. EFFECTORS that carry out either:
   
   • cell cycle checkpoints & DNA repair
   • senscence & apoptosis

Question 2

What are the 2 outcomes of the DDR?

Answer 2

1. Cell cycle checkpoints: temporarily arrests development, checking cell is ready to move on to the next stage - provides time for DNA damage to be repaired.
2. If DNA damage levels are too high or persist, cell undergoes:
   • apoptosis (cell death)
   • senescence (permanent cell cycle arrest)

Question 3

What are the 4 main DNA repair pathways used?

Answer 3

1. Base excision repair (BER)
2. Mismatch repair (MMR)
3. Nucleotide excision repair (NER)
4. Recombinatorial repair

Type of pathway used depends on type of damage and cell cycle phase.

Question 4

When is base-excision repair used?

Answer 4

Only removes 1 incorrect base so used in single nucleotide errors, e.g.

• uracil
• abasic site
• 8-oxoguanine
• SSB

Question 5

Describe the process of BER.

Answer 5

1. DNA glycosylases recognise specific types of base alterations via base flipping and catalyse the hydrolysis removal of the base from its sugar.
2. AP endonuclease cuts the phopshodiester backbone and deoxyribophosphodiesterase removes the phosphate sugar.
3. The single nucleotide gap is correctly filled by DNA polymerase.
4. DNA ligase seals the nick.

Question 6

When is mismatch repair used?

Answer 6

Repairs errors in DNA replication, e.g.

• A-G mismatch
• T-C mismatch
• insertion
• deletion

Question 7

Describe the process of MMR.

Answer 7

Removes multiple bases:

1. MutS recognises base misincorporation of a base during DNA replication.
2. Endonuclease nicks strand with incorrect nucleotide.
3. Exonuclease removes error-containing DNA segment (up to 2000 bases removed).
4. DPol III fills the gap and DNA ligase seals the nick.

Question 8

How does the endonuclease recognised which DNA strand is wrong during MMR?

Answer 8

Mature DNA is methylated at distinct sites whilst newer strand isn’t yet - newer strand assumed to be in error.

Question 9

When is nucleotide-excision repair used?

Answer 9

Removal of a section around damaged nucleotide(s), e.g.

• bulky adducts
• pyrimidine dimers
• intrastrand crosslinks

Question 10

Which components are involved in NER?

Answer 10

• Damage-sensing proteins: constantly scan genome and recognise helix distortions. Initiate repair when RNAP stalls at DNA lesion.
• Endonucleases: cleave DNA on either side of the damage and remove 30 nucleotides.

Question 11

When is recombinatorial repair used?

Answer 11

• Interstrand cross-link

- DSB

Question 12

What are the 2 types of recombinatorial (DSB) repair?

Answer 12

Non-homologous end-joining

Homologous end-joining

Question 13

How might non-homologous recombination lead to disease?

Answer 13

Segments on either side of the break are simply ligated - loss of nucleotides from degradation of ends.

Question 14

What is homologous end-joining initiated by?

Answer 14

DSB end resection - creates ssDNA overhang

Question 15

What does homologous end-joining use to fill the break?

Answer 15

Homologous template (preferably sister chromatid)

Question 16

What are the 2 types of process used to repair DSBs after resection and strand invasion?

Answer 16

1. Synthesis-dependent strand annealing (SDSA)
   - Causes loss of original sequence as homologous chromosome, with different sequence, is used to synthesise new DNA.
2. Double-strand break repair (DSBR)
   - Requires resolution of Holliday junctions (2 DNA strands switch partners between 2 double helices).
   - Produces hybrid chromosomes that contain genetic info from both homologs.

Question 17

What happens to the DDR in tumours?

Answer 17

Must be altered or suppressed to allow cancer development despite high levels of DNA damage and genomic instability.

Question 18

Give an example of DNA repair defects leading to cancer predisposition syndromes.

Answer 18

MMR defects can lead to Lynch syndrome and colorectal cancer.
Involve germline mutations in MMR genes and MSI-H (high-frequency microsatellite instability).

Question 19

Which important cell-cycle regulator is often mutated in tumour cells? What is its role?

Answer 19

• p53
• Role in tumour suppression by:
  
  • Promoting: senescence, apoptosis, autophagy, cell cycle inhibition, DNA repair.
  • Inhibiting: metabolic reprogramming, stemness and invasion & metastasis.

Question 20

What do DDR defects promote in terms of cancer cell sub-populations?

Answer 20

Promote genomic instability and mutation - drive tumour heterogeneity and evolution.

Question 21

What does the development of a heterogenous phenotype in tumours cause?

Answer 21

Therapy resistance

Metastasis

Question 22

How can we use DNA damage/replication stress in therapy?

Answer 22

Many cancer therapy agents act by causing DNA damage or replication stress.
E.g. Topotecan & irinotecan block resolution of I-DNA adducts during replication - persistent ssDNA breaks - dsDNA breaks when reached by replication fork.

Question 23

What are the problems associated with using DNA damage as an anti-cancer therapy?

Answer 23

• Many side effects (e.g. On other replicating cells):
  
  • Radiation - skin problems, fatigue, heart problems, etc.
  • Chemotherapy - hair loss, fertility problems, nausea, etc.
• Can promote tumour evolution and secondary cancers