13. Mechanisms of mutation in DNA & 16. Origins of other structural abnormalities

Question 1

What are the 3 main mechanisms of mutation?

Answer 1

1. DNA damage - due to endogenous and exogenous agents
2. Deficiency in DNA replication - variants escape 3’-5’ exonuclease proof-reading enzyme
3. Defects in DNA repair - closely tied to cell cycle. Failure at checkpoint can lead to accumulation of DNA damage

Question 2

What is the role of BER?

Answer 2

Corrects damage from oxidation, deamination, alkylation

Protects against ageing, neurodegeneration, cancer - e.g. MUTYH

Question 3

Give an example of a gene involved in BER

Answer 3

MUTYH - DNA glycosylase that recognises and removes damaged base, fixed by short (1 base) or long (>2 bases) patch pathway enzymes

Question 4

What is the role of NER?

Answer 4

Removes pyrimidine dimers / ICLs caused by UV radiation

Question 5

Give an example of a gene involved in NER

Answer 5

Mutations in NER protein complex cause Cockayne syndrome and xeroderma pigmentosa

Question 6

What is the role of MMR?

Answer 6

MMR proteins repair mismatched bases incorporated during DNA replication

Question 7

Give an example of a gene involved in MMR

Answer 7

MSH2-MSH6 or MSH2-MSH3 heterodimers recruit MLH1-PMS2 heterodimer - mismatch excised, gap filled by DNA pol, strand sealed by DNA ligase

Question 8

What does defective MMR cause?

Answer 8

Decrease in apoptosis, increase in cell survival –> selective growth advantage to cell –> cancer

Question 9

How is MMR deficiency often observed?

Answer 9

Microsatellite instability caused by replication slippage - alternation in length of tandem repeats due to lack of MMR

Question 10

Why are DSBs significant?

By what two methods are they repaired?

Answer 10

Can lead to translocations and creation of fusion gene

Homologous recombination repair (HRR) and Non-homologous repair (NHR)

Question 11

How does homologous recombination repair work?

Answer 11

Repairs DSBs using sister chromatid during G2 phase (after chromosome duplication)

Single strand of sister chromatid acts as template for repair

Facilitated by RAD51C, BRCA1/2, NBS, BLM

Question 12

What are the different types of non-homologous repair?

Answer 12

1. Non-replicative mechanisms:
   a) Non-homologous end joining (NHEJ)
   b) breakage-fusion-bridge cycle
   c) microhomology-mediated end joining (MMEJ)
2. Replicative mechanisms
   a) Fork stalling and template switching (FoSTes)
   b) Microhomology-mediated break induced replication (MMBIR)

Question 13

What are the 3 main types of DNA repair?

Answer 13

1. Direct reversal of DNA damage by enzyme, e.g. MGMT
2. Excision repair - recognition of damaged site
3. DSB repair

Question 14

What are the 3 excision repair mechanisms?

Answer 14

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

Question 15

How do chromosomal rearrangements occur?

Answer 15

Most recurrent rearrangements by NAHR between LCRs

Non-recurrent and those associated with translocations in leukaemia due to incorrect repair by DNA repair mechanisms

Question 16

How does NHEJ work?

Answer 16

No homology required, leads to 1-4bp deletion/insertion

Ku70/80 proteins recognise DSB and form scaffold to hold ends together

Artemis enzyme trims overhanging ends, DNA pol fills gap, DNA ligase joins ends

Question 17

What is breakage-fusion-bridge cycle?

What does it have a major role in?

Answer 17

Fusion of chromosome due to telomere erosion + unrepaired DSB –> creates dicentric chromosome

During anaphase, centromeres separated –> breakage of chromosome

Cycle only stops when chromosome acquires a telomere

Repeated cycles causes rearrangements

Major role in amplification in cancer

Question 18

How does FoSTeS work?

Answer 18

Fork stalling caused by formation of secondary structures

3’ end of lagging strand disengages from template and anneals to ssDNA in nearby replication fork where synthesis re-starts - causes dels/dups

Question 19

What is MMBIR?

How does it work?

Answer 19

Microhomology-mediated break-induced replication

Associated with restart of collapsed replication fork, initiated by ssDNA at DSB - 3’ end anneals to any ss homologous template - break repaired during replication

Question 20

What type of mutation does NHEJ often cause?

Answer 20

Translocations in cancer

Question 21

How can DSBs be repaired?

Answer 21

1. NHEJ
2. MMEJ
3. HRR

Question 22

How does MMEJ work?

Answer 22

Associated with deletions/insertions that flank DSB

Require shot region of homology either side of DSB

Question 23

What are the three main non-replicative, non-homologous repair mechanisms?

Answer 23

1. NHEJ
2. MMEJ
3. Breakage-fusion-bridge cycle

Question 24

What are the two main replicative, non-homologous repair mechanisms?

Answer 24

1. FoSTes
2. MMBIR

Question 25

What is the most common recurrent translocation & how does it occur?

What risk is associated with the translocation?

Answer 25

t(11;22)(q23.3;q11.2) - mediated by palindromic AT-rich repeats susceptible to DSBs

NHEJ between the 2 regions forms the translocation

Balanced carriers are normal, risk of Emanuel syndrome in offspring due to der(22)