Lecture 7 - DNA damage and Repair Flashcards
Give examples of physical mutagens.
Ultraviolet (UV) radiation is part of the electromagnetic spectrum emitted by the sun.
* UVC rays are absorbed by atmospheric o-zone
* 10% of UVB and most UVA rays reach the Earth’s surface
Both UVA and UVB are of major importance to human health. DNA absorbs UV radiation with a peak absorbance at 254 nm (absorbance of UV radiation results in DNA damage.
UV radiation promotes the formation of intra-strand cross-linked pyrimidine dimers.
* Linked Pyrimidines are not accommodated in active site of DNA polymerases and therefore it is replicated by low-fidelity TLS polymerases which introduces mismatches leading to mutation
Ionising radiation (X-rays, gamma rays) short wavelength, high energy radiation
Natural sources include therapeutic, diagnostic or occupational sources.
* 35% of DNA damage results from direct interaction of radiation energy with DNA
* 65% of DNA damage occurs indirectly as a result of attack by reactive oxygen species formed by ionisation of cellular water
This damages bases and breaks polynucleotide strand phosphodiester backbone - the strand breaks can have lethal effects particularly double strand breaks.
What is the ames test?
The Ames test for assaying the potential mutagenicity of chemicals
Many carcinogens are mutagens, the Ames test is a test for mutagens that is easier, cheaper and more ethically acceptable than animal testing.
1. Bacterial culture requiring histidine to grow is added to a defined minimal media with no histidine present.
2. The culture is then placed onto two agar plates one with the suspected mutagen present one without
3. The number of colonies arising from either spontaneous revertants (no mutagen) or colonies of revertants induced by the mutagen can be compared to see if the suspected mutagen has an effect.
Since some chemicals are converted to mutagens by liver enzymes, potential mutagens are treated with a mixture of liver enzymes prior to addition to the medium.
How do cells repair DNA damage?
Excision of Damaged DNA
1. Mismatch repair
2. Base excision repair
3. Nucleotide excision repair
Direct reversal of DNA damage
1. Repair of O6-alkylguanine
2. Enzymatic photoreactivation
Describe the process of mismatch repair.
Mismatch repair corrects mistakes made during replication, using the parental strand as a template
A mismatch repair system must:
1. Recognise mismatched base pairs
2. Discriminate between the correct (parental strand) base and the incorrect (daughter strand base in a mismatched pair.
3. Excise the incorrect base and carry out repair synthesis
The MutSLH mismatch repair system operates in bacteria - MutS and MutL proteins are highly conserved.
1. The Mut S protein is specifically recruited to mismatch bp in newly replicated DNA 2. Mut L is recruited to the site of the mismatch via interaction with Mut S 3. Mut H recognises the new DNA as it is not methylated (parent strand is methylated after replication) 4. The second lobe of Mut L interacts with Mut H at hemi-methylated GATC site, leading to the formation of the Mut SLH complex 5. Mut H nicks the new strand 6. The nicked strand is co-ordinately unwound by uvrD helicase and digested by an exonuclease. The exonuclease could be a 5'-3' o r3' to 5' exonuclease depending on which side the Mut H is located. 7. DNA is resynthesized by DNA pol III
Mismatch repair system can correct replicative insertions/deletions
* Repetitive regions of DNA can form hairpins on the template strand and be skipped during replication. This results in a deletion.
* Similar process in which hairpin forms on new strand can insert repeats
* Mismatch repair can detect and repair hairpins - the newly synthesised DNA is degraded, the hairpin unfolds and the new strand can be remade
* Defects in mismatch repair lead to increased rates of spontaneous mutation and cancer.
Describe base excision repair
Base Excision repair (Thomas Lindahl)
Cellular glycosylases with specificity for a particular type of damage have a key role in repairing endogenous DNA damage. The failsafe glycosylase is T:G mismatch glycosylase
1. Recognition of damaged base by a DNA glycosylase
2. Removal of damaged base by DNA glycosylase - DNA glycosylase cleaves between damaged base and sugar to leave abasic site
3. Recognition of the abasic site by the AP endonuclease - abasic sites are repaired by apurinic/apyrimidinic endonuclease (AP endonucleases)
4. Cleavage of the phosphodiester bonds flanking the abasic site - AP endonucleases cleave the backbone
5. Replacement of excised nucleotide by DNA polymerase and ligase - Gap is filled by DNA polymerase and ligated
Aziz Sancar and Tomas Lindahl found that the specificity is for helix distortion rather than specific type of damage
In bacteria, UvrA and UvrB scan DNA for distorted regions then UvrB (a helicase) unwinds the damaged region
UvrC nicks the damaged DNA, damaged DNA is then removed by UvrD, and DNA is resynthesized from the undamaged strand.
Describe nuclotide excison repair proteins
All organisms have a 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)
* TR removes stalled RNA polymerases
NER is mechanistically conserved in eukaryotes
* 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
* Skin cancer directly correlated to lack of repair of UV damage
Describe repair of alkylation damage.
- Alkylation on guanine (light blue) or the DNA backbone (dark blue) is repaired by alkyltransferase 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
AlkA removes methylated guanine bases during the first step of base excision repair.
Descrive enzymetic photoreactivation.
Direct reversal of DNA damage - Enzymatic photoreactivation
* 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
