L6: DNA damage and mutation

Question 1

DNA damage definition with examples

Answer 1

A change to the regular chemical structure of the DNA double helix
e.g…
- Break in the phosphodiester bb of poly-nt chain
- Loss of a base from the deoxyribose sugar
- Alteration to the structure of a base
- Non-complementary bases in the double helix (mismatched bps)

Question 2

Mutation definition w/ 2 examples

Answer 2

A permanent heritable change in the sequence of an organism’s genome
- Point mutation (alteration, insertion or deletion of one or a few bases)
- Chr. mutations (rearrangement, deletion, insertions)

Question 3

Define forward mutation

Answer 3

Wild type ‘active’ to mutant ‘defective’

Question 4

Define reversion mutation

Answer 4

Mutant ‘defective’ to wild type ‘active’ (can be either full or partial ie. sequence restored to a different aa that fully or partially restores function)

Question 5

Suppressor mutation definition

Answer 5

Changes seq. at a different location from original mutation in a way that compensates for original mutation
Either intergenic or intragenic

Question 6

Spontaneous mutation rate in different organisms

Answer 6

Euk…
- Germline nt substitution 10^-8 /nt/generation
- Somatic mutation rate is higher and varies in different tissues
Prok…
- 10^-9/nt/generation

Question 7

Lamarckism

Answer 7

The theory that mutation is ADAPTIVE, and organisms ‘direct’ mutations to adapt to a particular environment (conversely, mutation has been proven to be RANDOM)

Question 8

Types of premutagenic damage (replication of which causes spontaneous mutation)

Answer 8

DNA replication errors…
- Nt or template tautomerism causing mismatches
- Looping-out errors
- Replication slippage
Endogenous DNA damage…
- Base deamination
- Base loss
- Base modification as a result of exposure to metabolic products

Question 9

Mechanisms for fidelity of DNA replication in order of decreasing error frequency + overall rate of mis-incorporated nts not repaired

Answer 9

• Base pairing
• DNA pols (base selection, 3’ to 5’ proofreading exonuclease)
• Accessory proteins (SSB)
• Post replication mismatch repair

~1 in 10^9

Question 10

Effect of transient tautomerisation on genome

Answer 10

• Mismatches can be introduced as a result of base tautomerisation
• Tautomers in either strand can introduce non Watson-Crick base pairing -> mismatch
• If not repaired, error becomes fixed, mutation will arise -> premutagenic lesion

Question 11

Types of endogenous damage in detail x4

Answer 11

• Depurination of G: Leaves ‘apurinic’ site (AP site(, w/ no base attached to deoxyribose) -> premutagenic lesion
• Deamination of cytosine: Results in uracil -> permutagenic lesion, 5-MeC -> T, GT bp
• Alkylation: Alkyl grps may be added to several different pstns on bases by endogenous alkyl donors (may affect base pairing properties of base)
  e.g. S-adenosyl methionine
• Oxidative DNA damage: attack by reactive oxygen species (O2-, H2O2 etc.). 2300 bases damaged/cell/hr. Accounts for high mutation rate in mit. genomes. Damaged bases may be mutagenic or lead to strand breakage/replication block

Question 12

Examples of chemical vs physical mutagens

Answer 12

Chemical…
- Base analogs
- Base modifying agents (inc. alkylating agents)
- Intercalating agents
Physical…
- IR
- UV

Question 13

Examples of chemical mutagens (Base analog, 3 base modifiers)

Answer 13

• 5BU induces transition mutations (base analog)
• Nitrous acid deaminates (induces transition in C or A, also deaminates G but won’t affect base pairing) (base modifying agent)
• Hydroxylamine hydroxylates C inducing transition (bma)
• Methylmethane sulfonate/MMS methylates (e.g. G, inducing transition) (bma)

Question 14

Effect of intercalating agents on DNA

Answer 14

• Intercalating agents (e.g. ethidium bromide) have a flat, plaar structure, insert into minor groove resulting in partial unwinding
• Leads to insertions and deletions on replication