Flashcards in 18.01.13 Mechanisms of mutation in DNA Deck (18)
What is a mutation?
Permanent alteration in the DNA sequence
How do mutations arise?
Result of DNA damage, errors in replication or recombination and failure to repair DNA damage.
What factors can cause DNA damage?
1) Internal chemical events: depurination deamination and oxidative damage
2) Environmental agents: mutagenic chemicals (e.g. tobacco) and certain types of radiation (e.g. UV)
Give three examples of DNA lesions that require repair.
1) Missing damage: depurination (removal of purine) resulting from cleavage ofglycosidic bond between deoxyribose and the base by acid and heat. If left unrepaired, apurinic sites generate mutations during dna replication because specified paired base cannot be found.
2) Altered base: ionising radiation, alkylating, oxidising agents. Uncorrected deamination (C-->U) leads to a substitution even - adenine added instead of guanine.
3) Linked pyrimidines - caused by UV irradiation, usually thymine dimers. Distort DNA structure and intriduce bends/kinks that impede transcription and replication
4) single or double stranded breaks
5) Covalent linkage of strans e.g. by mitomycin C
6) Bulge in DNA due to a deletion of insertion of a nucleotide at recombination or replication
Which enzyme 'proofreads' DNA?
Recognises and corrects many errors made during replication.
What is the estimate mutation rate in humans?
1x10-4 --> 10x-6
Give three examples of DNA repair mechanisms.
1) Direct reversal of DNA damage
2) Excision repair - a) base excision repair b) nucleotide excision repair c) mismatch repair
3) Double stranded break repair a)homologous recombination repair b) non-homologous recombination
What is involved in the direct reversal of DNA damage DNA repair mechanism?
Damage can be repaired by specialised enzymes without the requirement for excision.
e.g. methylation of G creates O6-methylguanine which pairs Thymine instead of Cytosine. This can be repaired by O6-methylguanine methyltransferase (MGMT) that transfers the CH3 to a C in its active site restoring the guanine.
What are the different types of excision repair?
1) Base excision repair (BER)
2) Nucleotide excision repair (NER)
3) Mismatch repair (MMR)
What is the BER mechanism used for? What does this protect the cells from.
Removes oxidative damage
DNA glycosylases recognises and removes damaged bases by cleaving bond between base and deozyribose.
Free base released leaving a apurinic/apyrimidic site
BER protects against ageing, neurodegeneration and cancer. BER defects not common on cancer, but homozygous defects in MUTYH repair associated with colon cancer.
What are the two pathways of BER to repair a apurinic/apyrimidic site?
1) Short patch - involves a dingle nucleotide insertion
EP nuclease cleaves the phosphodiester bond 5' to the AP site. Removal of 5'-sugar-phosphate moeity creates a single nucleotide gap filled by DNApolymerase and sealed by DNA ligase.
2) Long patch - involves resynthesis of a patch 2-13 nt long.
What does nucleotide excision repair (NER) do? What are the four stages?
Removes pyrimidine dimers caused by UV radiation.
Complex process involving more than 30 proteins to remove fragments of ~30nt.
1) Detection of damage
2) Nuclease excision of the section of DNA the includes and surrounds the error
3) Filling in of the resulting gap by DNA polymerase
4) Sealing the nick between newly synthesised and older DNA
What syndromes are associated with inborn defects in NER?
Describe mismatch repair.
Recognises mismatched bases incorporated in DNA replication. Can be initated by:
1) MutSa - recognises mismatched base pairs and small indels.
2) MutSb - binds mainly to larger looped out insertion/deletion.
Both MutSa and b recruit the MusL complex. Mismatched bases are excised. DNA polymerase fills the gap, stands are sealed with DNA ligase.
What is the effect of lost MMR?
Decrease in apoptosis, increased cell survival and a potential increase in damage-induced mutagenesis.
Selective growth advantage to some cells and increased susceptibility to tissue-specific cancer.
Defective MMR can lead to replication slippage - frequently observed in microsatellite instability, implicated in most human cancers.
What disease are associated with defects in MMR genes?
What are the main mechanisms of double strand break repair?
1) Homologous recombination repair
2) Non-homologous recombination repair