BIOL253: Genetics L6:

DNA Damage and Mutation

DNA DAMAGE = change to regular chemical structure of DNA double helix e.g.,
• Break in phosphodiester backbone of polynucleotide chain.
• Loss of base from deoxyribose sugar.
• Alteration to structure of a base.
• Non-complementary bases in the double-helix (mismatched base pairs)

DNA damage and mutation are NOT the same thing.

DNA damage can be detected and repaired.

DNA damage often leads to mutation if damaged DNA is replicated.

MUTATION = permanent heritable change in the sequence of an organism’s genome.

• Point mutation – alteration, insertion, or deletion or one or a few bases at a time.
• chromosome mutations – rearrangement (translocation), deletion, insertions.
 Larger changes compared to point mutation.
Mutation but DNA is ‘normal’ – not DNA damage as just changes sequence of bases.
Mutations cannot be recognised and repaired like normal damaged DNA.

POINT MUTATIONS
Classifications of base-pair substitution mutations
• Transition – mutation where purine is replaced with another purine, on the complementary strand the
pyridine is replaced by another pyrimidine.

• Transversion – pyrimidine replaced by purine and vice versa.

• Missense – single base pair substitution alters codon, codes for a different amino acid.

• Nonsense – nucleotide change from coding codon to stop codon. Stops translation of polypeptide
sequence (TAA< TAG, TGA = stop codons)

Types of base-pair substitution mutations

How much of an effect a change has on the protein?
• Neutral – change from amino acid to another amino acid with similar chemical properties.
• Silent – change in codon, but codes for same amino acid.
• Frame shift – addition/deletion one/few base pairs leads to change in reading frames. Each
subsequent codon is affected.
 Nonsense and frameshift have the greatest effect on proteins.
BIOL253: Genetics L6:
DNA Damage and Mutation

Forward, Reverse and Suppressor Mutations

• Forward mutations – wild type ‘active’ to mutant ‘defective’ gene.
• Reserve (reversion) mutations – mutant ‘defective’ to wild-type ‘active’ gene.
- True reversion = restores sequence to code for the wild type amino acid in affected protein.
- Partial reversion = changes sequence at site of original mutation to some other amino acid that
fully or partially restores protein function.
• Suppressor mutation – changes sequence at different location from original mutation which
compensates for original mutation.
- Intragenic = within the same gene.
- Intergenic = within a different gene. (When genes act in a complex)

Most mutations are spontaneous

 Arise without exposure to exogenous agents.
Spontaneous mutation rate:
Eukaryotes:
• Hard to measure, varies across different sequence classes.
• Germline nucleotide substitution 10-8/nt/generation.
• (~30 new mutations in 3Gb haploid genome inherited from each parent)
• Somatic mutation rate is higher and varies in different tissues.
Prokaryotes:
• ~10-9/nt/generation (or ~10-6/typical 1kb gene/generation) – i.e., for a 1kb gene 1 mutation in ~1 million
cell divisions. (1Kb = size of typical prokaryotic gene)

Mutations are random – NOT adaptive

 Most are problematic.
• Adaptive – organisms’ ‘direct’ mutations to adapt to a particular environment (Lamarckism)
• Random – changes happen by chance, sometimes are adaptive.

Adaptive mutation - proposed if plated bacteria onto plates with T1 phage – get number of bacteria that
became resistant to T1 at a specific rate. Occurs due to bacteria adapting to exposure to T1.
Random mutation – proposed resistance of bacteria depends on of there are any pre-existing mutations to T1.
Number of resistant bacteria would vary depending on bacteria on plate.
 Observation that T1 phage resistance arises could support either theory.

HOW DO SPONTANEOUS MUTATIONS ARISE?

 Occur because of replication of DNA containing premutagenic damage caused by:
DNA replication errors:
• Nucleotide or template tautomerism causing mismatches.
• Replication slippage (template/new strand loops out to allow for deletion of extra bases)
Endogenous DNA damage: (caused by things inside the cell)
BIOL253: Genetics L6:
DNA Damage and Mutation
• Base deamination.
• Base loss.
• Base modification because of exposure to metabolic products.

DNA replication – mechanisms ensure fidelity of DNA synthesis

Mechanism Cumulative Error Frequency
Base pairing ~10-1-10-2 (1 in 10-100)

DNA polymerase (base selection, 3’-6’ proofreading exonuclease) ~10-5-10-6 (1 in 105-106)

Accessory proteins (SSB) ~ 10-7 (1 in 107)

(binds ssDNA once unwound)
Post replication – mismatch repair ~ 10-9 -10-10 (1 in 109-1010)
Overall rate of mis incorporated nucleotides not repaired is 1 in 10 -1010
9

Why does DNA replication need to be faithful?

 Mismatches from replication errors can become fixed
(permanent) as mutations following a further round of DNA
replication.
• Mismatches = premutagenic lesions. Not yet a
mutation.
• Can be recognised as different to normal DNA (not
normal base pair).
• If replicated before it can be repaired =
mutation.
• DNA is no longer mismatched but incorrect DNA
sequence on both strands, looks like normal DNA
so won’t be picked up.
• Mutations likely to disrupt protein function,
occasionally confer advantage.

REPLICATION OF RARE TAUTOMER’S CAN LEAD TO MUTATION

• Mismatches can be introduced because of base tautomerisation
• Bases adopt rare tautomeric form, either in template strand, or in incoming nucleotide within DNA
polymerase active site. (only important if happens during replication)
• Tautomers form non-Watson-Crick base-pairs, so incorrect nucleotide incorporated, resulting in
mismatch (DNA damage)
• If mismatch not repaired, after next round of replication, permanent sequence change (i.e. mutation) will
arise.
BIOL253: Genetics L6:
DNA Damage and Mutation

Spontaneous generation of addition and deletion mutants by DNA looping-out errors during replication:
• new or template strand.
• Typically in repeat sequence.
• Copying of base is missed or
additional base added.
• When DNA is copies – the
base that is missed is deleted.
• DNA damage can be
recognised – needs to repeat
again for it to become a
mutation.

Endogenous Damage: Hydrolysis – DEPURINATION

• Depurination of Guanine (bone between sugar and base broken – releasing G) – leaves APURINIC site
(AP site) with no base attached to deoxyribose - premutagenic lesion. (Happens by reaction with
water - hydrolysation)
• Mammalian cells lose 18000 bases per cell per day.
• Purines hydrolysed more the pyrimidines.
• Depurination>>Depyrimidination.
• AP sites result in random base substitution or base-skipping during replication – leads to
substitution/deletion.
• Further round of replication leads to mutation.

Endogenous Damage: Hydrolysis – DEAMINATION

• Deamination (removal of amino group) of cytosine results in uracil – premutagenic lesion/DNA
damage – uracil not usually present in DNA. If replicated = mutation.
• Eukaryotes can methylate cytosine – deamination of this leads to thymine (normal in DNA) but normally
is not matched to C. DNA damage so may be corrected – if replicated = mutation.
• 5-MeC ->T
• GT base pair – premutagenic lesion
• CpG sequences Mutation ‘hotspot’
• 100-500 bases
deaminated per cell/day
BIOL253: Genetics L6:
DNA Damage and Mutation

Endogenous Damage: ALKYLATION

• Alkyl (methyl/ethyl) groups may be added to several different
positions on bases by endogenous alkyl donors.
• Some groups are very reactive.
• Guanine can be methylated on the O6 position due to exposure to
endogenous metabolites.
• Base modification may affect base-pairing properties of modified
base.
• DNA damage- can be repaired – if replicated = mutation.

Endogenous damage: OXIDATIVE DNA DAMAGE – leads to spontaneous mutation

• Attack by reactive oxygen species (ROS) O2-, H2O2 , OH▪ on e.g. guanine
• Premutagenic lesion
• Seen especially in the mitochondrial genome (can leak from the electron transport chain).
• Guanine is susceptible to oxidative damage – doesn’t insert Cytosine, inserts Adenine.

Mutations can be induced by exposure (deliberate or accidental) to mutagens

Chemical mutagens:
1. Base analogues.
2. Base modifying agents (including alkylating agents)
3. Intercalating agents.
Physical mutagens:
1. Ionising agents.
2. Ultraviolet (UV) radiation.
BIOL253: Genetics L6:
DNA Damage and Mutation

BASE ANALOGUES
5-bromouracil (5BU) induces transition mutations.
^ Can be used to induce mutations.

ACTION OF BASE-MODIFYING AGENTS

a. Nitrous acid – deaminates

b. Hyxdroylamine – hydroxylates
Can change properties of bases – see
effects after replication that lead to
mutation.
c. Methylmethane sulfonate (MMS) –
methylates
Methylates range of bases. Different
effects depending on different bases.
BIOL253: Genetics L6:
DNA Damage and Mutation
Can completely block replication or cause mutations.

Susceptibility of guanine to DNA damage

• Guanine is particularly reactive – can be affected by oxidative damage.
• All impact how it base pairs and whether it can be replicated at all.

INTERCALATING AGENTS INDUCE FRAME-SHIFT MUTATIONS

• Intercalating agents e.g. ethidium bromide, have flat, planar structures that insert in the minor groove of
the helix, resulting in partial unwinding.
• Bind between the stacked base pairs.
• Leads to insertions and deletions on replication. (frame shift mutations)

Chemical mutagens in the environment: Chemicals

metabolised to functional alkylating agents
• Found in environment.
• Direct or can be converted to be mutagenic in
organisms.
• Procarcinogen – converted into active
carcinogen.
• Can attach to DNA complex.

Benzopyrene sources: smoking, car fumes, processed meat.