BIOL253: Genetics L7: DNA damage, mutation, repair & damage tolerance

DNA DAMAGE, MUTATION, DNA REPAIR, DNA DAMAGE TOLERANCE

Physical Mutagens: UV radiation

• UV radiation is part of electromagnetic spectrum emitted by sun.
(200-400nm)
• UVC rays are absorbed by atmospheric ozone.
• 10% of UVB rays and most UVA rays reach Earth’s surface.
• UVA & UVB are of major importance to human health.
• DNA absorbs UV radiation with a peak of absorbance at 254 nm.
• Absorbance of UV radiation results in DNA damage.
• Note: X-rays and gamma rays have a shorter wavelength, higher
energy radiation – damaging effects on DNA.

UV radiation promotes the formation of intrastrand cross-linked pyrimidine dimers

• If UV radiation reaches organisms – directly absorbed by DNA in skin.
• Causes damage where there are adjacent pyrimidine residues in the same strand (T, C).
• 2 types of DNA damage:
- Links between adjacent bases (5,5 bond and 6,6, bond) ~75% make cyclobutane pyrimidase
products.
- Links between 6,4 position of adjacent pyrimidines ~25% form 6,4 photoproduct.
• Helix becomes distorted and partially unwound due to the links.
• Linked pyrimidines – not accommodated in active site of replicative DNA polymerases (DNA polIII, DNA
pol d, DNA pol e)
• Cannot replicate DNA when comes across cyclobutene or photoproduct.
• Replicated by low-fidelity TLS polymerases (more relaxed active site) accommodates and replicates
damaged DNA – introduces mismatches – leads to mutation.

Physical mutagens: Ionising radiation (X-rays, gamma rays) short wavelength, high energy radiation
• Natural sources (granite, radon – geology), therapeutic (cancer treatment), diagnostic (xray), or
occupational sources.
• 35% of DNA damage results from direct interaction of radiation energy with DNA.
• 65% of DNA damage occurs indirectly because of attack by reactive oxygen species (ROS) formed by
ionisation of cellular water.
• Damages bases.
• Breaks polynucleotide strand phosphodiester backbone (ss breaks and ds breaks)
• Lethal effects due to strand breaks - particularly double-strand breaks (DSBs)
BIOL253: Genetics L7: DNA damage, mutation, repair & damage tolerance
The Ames test for assaying the potential mutagenicity of chemicals
• Many carcinogens are mutagens.
• Ames test is test for mutagens. (Looking if bacteria can mutate)
• Easier/cheaper/more ethically acceptable than animal testing.
• Since some chemicals are converted to mutagens by liver enzymes,
potential mutagens are treated with a mixture of liver enzymes prior to
addition to medium.

• Salmonella is a histidine auxotroph (cannot synthesise histidine) – provide

with histidine to grow.
• Plate salmonella onto minimal media (lacks histidine) – cells cannot grow.
• Include chemical that might be a mutagen – incubate and look for growth
of colonies.
• Only grown if reversion mutation that allows then to synthesise histidine.

How do cells repair DNA damage?

Excision of damaged DNA
 Cutting out.
1. Mismatch repair (MMR)
2. Base excision repair (BER)
3. Nucleotide excision repair (NER)
Direct reversal of DNA damage (specific)
 Damage is converted back to normal.
1. Repair of O6-alkylguanine
2. Enzymatic photoreactivation

1. Excision of DNA damage – Mismatch Repair (MMR)

Corrects mistakes made during replication, using the parental strand as template. Conserved in eukaryotes.
Must:
• Recognise mismatched base pairs.
• Discriminate between correct (parental strand) and incorrect (daughter strand) base in mismatched
pair.
• Excise incorrect base, carry out repair synthesis.
MutSLH mismatch repair system operates in bacteria. (Mut = mutator)
MutS and MutL proteins are highly conserved (found in every organism) – they recognise the mismatched
base pairs.
MutH – involved in discrimination between correct and incorrect base in mismatch repair – not conserved in
eukaryotes 

Base can adopt rare tautomer – when reverts to normal – MMR system MutSLH must fix mismatch.
If the genes in bacteria are themselves mutated (don’t code for functional bacteria), then those strains that
have the defective mismatch repair proteins accumulate mutations in other genes at a higher rate.

Mismatch Repair
 Each of the Mut S, L and H genes encodes a protein that carry out MMR in bacteria.
1. MutS protein is specifically recruited to mismatch bp in newly replicated DNA via interaction with sliding
clamp.
Methylation of GATC sequences – in newly replicated DNA it is hemi-methylated with the parental
strand being methylated and daughter strand unmethylated. Basis for strand discrimination in bacteria.
2. MutS recruits MutL protein. MutL protein (2 lobe protein) is recruited to site of mismatch via interaction,
bound by its first lobe with MutS.
3. MutL protein interacts with MutH protein via its 2nd lobe. MutL recognises and binds to the hemi-
methylated GATC sequences (can be anywhere in the genome). MutL can loop DNA through it until it
reaches a MutH protein bound to a hemi-methylated DNA site.
BIOL253: Genetics L7: DNA damage, mutation, repair & damage tolerance
4. MutL binds the MutS bound to mismatch and the MutH bound to the hemi-methylated GATC. When this
complex is assembled – this activates the MutH protein to introduce a nick into the newly
polynucleotide strand at that GATC sequence.
5. Nicked strand is co-ordinately unwound (by UvrD helicase) and digested by an exonuclease.
• Exonuclease required is a 5’ to 3’ exonuclease, if MutH nicks at site that is 5’ to mismatch (RecJ
nuclease or exonuclease VII) (as in this case).
• Exonuclease required is a 3’ to 5’ exonuclease it MutH nicks at a site that is 3’ to mismatch
(exonuclease I).
6. DNA including the mismatch is degraded by exonuclease. This removes the newly synthesised strand
that includes the mismatch base pair. Once the mismatched base pair is reached, nucleolytic activity
van be terminated and 3’ hydroxyl left can be used to synthesise new DNA using parental
complimentary strand.
7. DNA is resynthesized by DNA pol lII (or DNA Pol I), which closes the gap by correcting the mismatch.

Mismatch repair system can correct replicative insertions/deletions

• Repetitive regions of DNA can form hairpins on the template strand (black) and be skipped during
replication in newly synthesised strand (green).
• If not repaired – replication leads to mutation.
• If recognised and loop strand identified as parental strand and removed – no mutation.
• Results in repeat contraction after the next replication round (blue)
• Similar process in which hairpin forms on new strand can insert repeats.
• Mismatch repair can detect and repair hairpins – the newly synthesized DNA is degraded, the hairpin
unfolds, and the new strand can be re-made.
• Defects in mismatch repair led to increased rates of spontaneous mutation (mutator phenotype) and
cancer. (some colorectal cancer associated with inheritance of defective mismatch repair gene –
cancers particularly have issue with mismatch repair of insertion/deletion loops)

2. Excision of DNA Damage – Base Excision Repair (BER)

 Acts to remove damaged bases – bases removed and excised in two-step mechanism - Recognise
particular type of damaged base and then remove damage.
BIOL253: Genetics L7: DNA damage, mutation, repair & damage tolerance
• Cellular glycosylases with specificity for particular type of damage. (Implies that type of damage is
significant!)
• 8-oxoguanine DNA glycosylase, Uracil-DNA glycosylase, 3-mA-DNA glycosylase I (2 of these, one is
very specific will remove 3-methyl adenine from DNA, the second is less specific, will remove different
methylated alkylated bases), Thymine glycol DNA glycosylase.
• Failsafe glycosylase – specifically recognises T:G mismatch
glycosylase. Occurs where 5 methyl cytosine is deaminated –
ends up as thymine, ending up with T-G mismatch.

• Damaged G base – recognise – recognition is specific to the type

of damage. If enzyme recognises the damaged base – good
indication that the damage occurs endogenously.
• BER – key role in repairing endogenous DNA damage.
• Glycosylases recognise damage and they flip the
damaged base out from being inside the double helix to
being outside the double helix.
• Then break glycosidic bond between the sugar and the
damaged base.
• Polynucleotide backbone is not incised, but the base is
removed by cleavage of the glycosidic bond.
• DNA glycosylase cleaves between damaged base and
sugar to leave abasic site (removed purine or
pyrimidine).
• Abasic sites are repaired by apurinic/apyrimidinic
endonuclease (AP endonucleases)
• AP endonucleases cleave the phosphodiester backbone
and bonds that are flanking the abasic site and replaces
excised nucleotide using DNA polymerase and ligase.
AP site. Seals the nick.

3. Excision of DNA Damage – Nucleotide Excision Repair (NER)

 Specificity for helix distortion rather than specific type of damage.
 Bulky lesions that distort the DNA helix are repaired by nucleotide
excision repair

• Don’t recognise a specific type of damage.

• Remove oligonucleotides (many nucleotides linked)
• General repair pathway – repairs damage that distorts the helix. E.g.,
UV damage.
• Repairs 6,4 photoproduces better than cyclobutene as it distorts the
helix more.
• Proteins originally designated as Uvr UUV repair) – studies in bacteria.
Graphs show broad specificity of NER.
- Red line – shows bacteria mutatino in one of the Uvr genes.
Exposed to mutating agents.
- Black line – survival of wildtype strain.
- Shows that exposure reduces survival and that Uvr mutants
are sensitive to agents.

Nucleotide Excision Repair

 Proteins are not conserved but mechanisms are in eukaryotes.
1. In bacteria, UvrA and UvrB proteins (in dimer) scan DNA for
distorted regions.
2. Then UvrB (a helicase) unwinds the damaged region. Allowing
release of the UvrA protein.
BIOL253: Genetics L7: DNA damage, mutation, repair & damage tolerance
3. UvrC recruited and nicks the damaged DNA that is bound to the UvrB. UvrC is an endonuclease that
acts only when it is associated with UvrB – by introducing nick 5’ and 3’ of the DNA damage. Results in
release of short oligonucleotide (approx length 13N).
4. Damaged DNA the oligonucleotide is then removed by UvrD.
5. DNA is resynthesized from the undamaged strand using the 3’ hydroxyl at end where damage was
removed can be used by DNA polymerase to fill the gap. Using info in undamaged DNA strand.

All organisms have nucleotide excision repair capacity

• Nucleotide excision repair (NER) proteins are not conserved between bacteria and eukaryotes – but
mechanism is conserved.
• NER operates anywhere in the genome (global genomic repair; GGR)
• Damage in genes that are actively transcribed is preferentially repaired (Transcription-coupled repair;
TCR).
• TCR removes stalled RNA polymerases.

NER is Mechanistically Converted in Eukaryotes

Eukaryotes have larger oligonucleotides.
• More complex, released oligonucleotide ~32mer.
• Defects in NER result in a mutator phenotype.
• Inherited NER defects cause Xeroderma Pigmentosum (XP) (and Cockayne syndrome).
• Rare, fatal, autosomal recessive disorder affects 1 in 250000 worldwide.
• Sun sensitive, predisposition to skin cancer.
• If keep out of the sun – have longer life span than they would.
• Skin cancer directly correlated to lack of repair of UV damage.

Direct reversal of DNA damage – Repair of Alkylation Damage

DNA can be alkylated by exposure to endogenous agents e.g., S-adenosyl
methionine. Alkylation can occur at multiple different positions of bases.

• Alkylation on guanine (Alkylated on O6 position) (light blue) or the DNA

backbone (dark blue) is repaired by alkyltransferases in the adaptive
response to alkylation damage.
• The E. coli alkyltransferase is Ada aka O6-Methylguanine-DNA
Methyltransferase.
• Alkyl group is transferred on to Ada and Ada is inactivated.
• Methyl-Ada stimulates production of more Ada, and the AlkA glycosylase
(broader substrate specificity – removes alkylation from all other bases).
• AlkA removes methylated guanine bases during first step of base excision
repair.

Direct reversal of DNA damage – Enzymatic photoreactivation of pyrimidine dimers photolyase enzyme
Repair damage caused by UV radiation. Uses photolyase. Very specific pathway.
Products of DNA damage sustained by UV radiation – either 6,4 photoproduct or cyclobutene pyrimidine
dimers are recognised in DNA, by DNA photolyase – which binds to the damaged DNA. On absorption of a
photon of visible light, can break apart those bonds that have been formed between adjacent pyrimidines in the
DNA.
• Found in all organisms (other than placental mammals).
• Photolyases may be specific for either CPDs or 6-4 PPs.
• Photolyases contains two noncovalently bound chromophores.
• An antenna pigment that absorbs sunlight.
• Catalytic cofactor – fully reduced Flavin-adenine dinucleotide (FADH-).
• Mechanism – electron transfer from FADH- to UV-induced lesion, dimer splitting, transfer of electron
back to FADH to generate
FADH-.