DNA Damage and Mutation

DNA damage

A physical or chemical alteration to DNA structure that deviates from its normal form.

Backbone damage

• Breakage of phosphodiester bonds

• Can produce:

  • Single-strand breaks (SSBs)

  • Double-strand breaks (DSBs) → highly lethal

Base loss

• Cleavage of N-glycosidic bond

• Leaves AP (apurinic/apyrimidinic) site

• No base → replication ambiguity

Base modification

• Chemical alteration of base structure

• Changes:

  • Hydrogen bonding

  • Base pairing specificity

Mismatched base pairs

• Non-Watson–Crick pairing

• Often arises during replication

Key properties of DNA damage

• Temporary

• Recognised by repair systems

• Repairable

• Consequence: premutagenic lesion

Mutation

A permanent, heritable alteration in nucleotide sequence

Key properties of mutation

• Permanent

• Non repairable

• Heritable

• Often from unrepaired DNA damage

Damage to mutation

• DNA damage occurs

  • e.g. cytosine → uracil

• Replication occurs before repair

  • DNA polymerase inserts base opposite damaged site

• Mispairing occurs

  • U pairs with A instead of G

• Second round of replication

  • A pairs with T → mutation fixed

Point mutations

Alterations affecting one or a few nucleotides

Types of point mutations

• base substitutions

• insertions / deletions (indels)

• large-scale mutations

base substitutions - transitions

• Purine ↔ Purine (A ↔ G)

• Pyrimidine ↔ Pyrimidine (C ↔ T)

• Often caused by:

  • Tautomerization

  • Deamination

  • Base analogues (e.g. 5BU)

Example: C → T (common at CpG sites)

base substituions - transversions

• Purine ↔ Pyrimidine

Mechanistic basis:

• Larger structural distortion

• Often caused by:

  • Oxidative damage

  • Chemical mutagens

Example: G → T (via oxidative damage)

insertions / deletions (indels)

Mechanism: replication slippage

Occurs in repetitive DNA sequences

Two possibilities:

1. Template strand loop-out → deletion in new strand

2. Newly synthesized strand loop-out → insertion

Frameshift mutations

• Occur if insertion/deletion ≠ multiple of 3

• Alters:

  • Reading frame

  • All downstream amino acids

  • Alters entire protein sequence downstream

  • Often introduces premature STOP

Large-scale mutations

Types:

• Deletions

• Insertions

• Translocations

• Inversions

Impact:

• Affect multiple genes

• Often severe phenotypic consequences

Synonymous (silent) mutations

Mechanism:

• Codon change does NOT alter amino acid

Reason:

• Degeneracy of genetic code

• Can subtly affect:

  • Translation efficiency

  • mRNA stability

non-synonymous mutations

• nonsense mutations

• missense mutations

  • conservative missense

  • non-conservative missense

nonsense mutations

• Codon → STOP codon (UAA, UAG, UGA)

Consequences:

• Premature termination

• Truncated protein

• Often:

  • Nonfunctional

  • Rapidly degraded

missense mutations

Codon → different amino acid

conservative missense

• Similar biochemical properties

Example:

• Lys → Arg (both basic)

Effect:

• Minimal structural disruption

non-conservative missense

• Different chemical class

Example:

• Lys → Thr (basic → polar)

Effect:

• Disrupts:

  • Folding

  • Active sites

  • Interactions

forward mutation

Wild type → mutant

reverse mutation

True reversion:

• Original sequence restored

Partial reversion:

• Different sequence → same/similar function

suppressor mutation

Second mutation compensates for first

Types:

• Intragenic

• Intergenic

adaptive theory (incorrect)

Claim:

• Mutations occur in response to environment

Problem:

• Implies directed evolution

random theory (correct)

Supported by Luria–Delbrück experiment

Key finding:

• Mutations occur before selection

• Distribution is random

KEY CONCEPT: Selection acts on mutations — it does NOT cause them

endogenous damage

Source: Internal cellular processes

• tautomerisation

• depurination

• deamination

• oxidative damage

• alkylation

• replication slippage

Includes:

• Replication errors

• Hydrolysis

• ROS

• Metabolic by-products

exogenous damage

Source: External environment

• base analogues (5BU)

• base-modifying agents

• intercalating agents

• UV radiation

• ionising radiation

Includes:

• Radiation

• Chemicals

• Pollutants

spontaneous mutations

Occur: Without external agents

Causes:

• Tautomerization

• Replication errors

• Endogenous damage

induced mutations

Caused by: Mutagens

Examples:

• UV light

• X-rays

• Chemical mutagens

tautomerisation

• Proton shifts → rare forms

Consequence:

• Non-standard base pairing

Example: A* pairs with C

depurination

• Hydrolysis of glycosidic bond

Rate: ~20,000/day in mammalian cells

Outcome:

• AP site

• Random nucleotide insertion

deamination

• C → U

• 5mC → T

Consequence:

• GC → AT transitions

Special case:

CpG islands = mutation hotspots

oxidative damage

Source: Reactive oxygen species (ROS)

Example: G → 8-oxoG

Consequence: Mispaired with A

alkylation

Addition of methyl/ethyl groups

Example:

O6-methylguanine → pairs with T

replication slippage

Occurs in: Repetitive sequences

Result: Insertions/deletions

base analogues (5BU)

• Mimics thymine

• Can pair with:

  • A (normal)

  • G (rare tautomer)

Result:

AT ↔ GC transitions

base-modifying agents

Nitrous acid: Deaminates bases

Hydroxylamine: Modifies cytosine

Alkylating agents: Add methyl groups

intercalating agents

• Insert between bases

Effect:

• DNA unwinding

• Insertions/deletions

UV radiation

• Forms pyrimidine dimers

Effect: Blocks polymerase

ionising radiation

Effects:

• DNA strand breaks

• Genome instability

DNA replication fidelity

• base pairing error rate = 10⁻¹–10⁻²

• DNA polymerase error rate = 10⁻⁵–10⁻⁶

• proofreading error rate = 10⁻⁷

• Mismatch repair error rate = 10⁻⁹–10⁻¹⁰

Ribonucleotide misincorporation

• Up to 1 in 625 bases

• Major source of lesions

mutation rates

• Prokaryotes: ~10⁻⁹/nt/generation

• Humans: ~30 mutations/genome/generation

Ultraviolet radiation

• Part of electromagnetic spectrum emitted by sun

• Both are biologically significant mutagens

• Shorter wavelength than visible light

• Higher energy

• Peak DNA absorption at 254 nm

• Absorption leads to photoexcitation of bases

• DNA bases (especially pyrimidines) absorb UV strongly

UVA

• longest wavelength

• Most Reaches Earth’s surface

• major impotence to human health

UVB

• ~10% reaches earth’s surface

• Most UVB absorbed by ozone layer

• major importance to human health

UVC

• All UVC rays absorbed by atmospheric oxygen

• Shortest wavelength

Formation of Pyrimidine Dimers as a result of UV damage

• UV energy is absorbed by adjacent pyrimidines:

  • Usually thymine–thymine (T–T)

  • Can also be C–T or C–C

• Covalent bonds form between adjacent bases:

  • Cyclobutane pyrimidine dimers (CPDs)

  • 6–4 photoproducts

• Result: Intrastrand cross-linking

Structural consequences of UV damage

• Bases become covalently linked

• DNA helix becomes:

  • Distorted

  • Locally unwound

• Prevents normal base pairing

Formation of intrastrand cross-linked pyrimidine dimers

• promoted by UV radiation

• Linked pyrimidines – not accommodated in active site of replicative DNA polymerases (DNA polIII, DNA pol d, DNA pol e)

• Replicated by low-fidelity TLS polymerases – introduces mismatches – leads to mutation

• Constrain two pyrimidines against each other – distorting

• Replicative polymerase find difficult to insert nucleotides opposite pyrimidines in the template strand

• Tls = translesion

UV radiation effect on DNA replication

Problem:

Replicative polymerases:

• Bacteria: DNA pol III

• Eukaryotes: DNA pol δ, DNA pol ε

➡ Cannot accommodate distorted template

Outcome:

• Replication stalls

• Fork progression blocked

Translesion Synthesis (TLS) — DAMAGE TOLERANCE

Solution: Cells recruit TLS polymerases

Properties of TLS polymerases:

• Can bypass DNA lesions

• Have:

  • Larger active sites

  • Reduced specificity

• Outcome: TLS polymerases introduce mismatches → mutations

Translesion Synthesis trade-offs in replicative polymerases

• high fidelity

• precise

• normal replication

Translesion Synthesis trade-offs in TLS polymerases

• low fidelity

• Error-prone

• Damage bypass

Mutation formation from UV damage

1. UV → pyrimidine dimer (DNA damage)

2. Replicative polymerase stalls

3. TLS polymerase bypasses lesion

4. Incorrect base inserted

5. Next replication → mutation fixed

Common mutation:

• C → T transition

• Especially at dipyrimidine sites

Ionising radiation (X-rays, g-rays)

•Damages bases (oxidative damage)

•Breaks polynucleotide strand phosphodiester backbone (ss breaks and ds breaks) 

• Lethal effects due to strand breaks - particularly double-strand breaks (DSBs)

•Radiolysis – react with DNA bases to bring about oxidative damage

•Ionising radiation passes through the DNA and causes clustered ionising events – double strand break – 2 ends free to dissociate from each other

Physical properties of ionising radiation

• Short wavelength

• Very high energy

• Capable of:

  • Ionising atoms

  • Breaking chemical bonds

Sources of ionising radiation

• Natural background radiation

• Medical imaging (X-rays)

• Radiotherapy

• Occupational exposure

Direct DNA Damage from ionising radiation

(~35%)

Radiation interacts directly with DNA

Effects:

• Breaks chemical bonds

• Damages:

  • Bases

  • Sugar-phosphate backbone

Indirect DNA damage from ionising radiation

(~65%)

Mechanism:

1. Radiation ionises water:

   • H₂O → free radicals

2. Generates reactive oxygen species (ROS):

   • OH•

   • H₂O₂

   • O₂⁻

   • Attack DNA bases

   • Cause oxidative damage

Base damage from ionising radiation

• Oxidation

• Chemical modification

Example: Guanine → oxidised derivatives

Single-Strand Breaks (SSBs) from ionising radiation

• Break in one strand

• Usually repairable

Double-Strand Breaks (DSBs) from ionising radiation

• Two closely spaced strand breaks

• Can arise from:

  • Direct radiation

  • Clustered ROS damage

Why DSBs are severe:

• No intact template strand

• Ends may:

  • Diffuse apart

  • Be incorrectly rejoined

Clustered DNA damaged from ionising radiation

• Ionising radiation produces clusters of ionisation events

Consequence:

• Multiple lesions in close proximity:

  • Base damage

  • Strand breaks

Result:

• Repair becomes difficult

• High probability of:

  • Mutation

  • Chromosomal rearrangements

Radiolysis of Water from ionising radiation

Radiation → water ionisation → ROS formation

Importance:

• Explains why:

  • 65% of damage is indirect

  • Even non-DNA-targeted radiation causes DNA damage

Mutation Formation from Ionising Radiation

A. Misrepair of base damage

→ point mutations

B. Misrepair of DSBs

• Deletions

• Translocations

• Chromosomal rearrangements

C. Replication across damaged DNA

→ misincorporation

Intercalators

• DNA distortion

• Frameshifts