L24: Mutation And DNA Repair In Bacteria Flashcards
Gene
The nucleic acid sequence that codes for polypeptide, tRNA or rRNA
Genotype
Collection of genes an organism has, or its genetic composition
Phenotype
Observable characteristics of an organism, or expression of genes
Mutation
Permanent, heritable change to base sequence of DNA
Wild type
Organism as it was first isolated from nature. Considered to be normal type
Mutant
Organism that differs from wild-type as result of mutation
Mutation of DNA
Rare
Main cause: errors in DNA replication (incorrect base inserted into daughter DNA strand)
- > detectable mutation rate at ~1 in 10^7 to 10^11 per bacterial cell for any particular gene
- > sensitive detection systems needed
DNA damaged by mutagens
DNA modifying agents: e.g. add alkyl groups to bases, change base pairing, such as ethylmethane sulphonate (EMS) which adds ethyl groups
Intercalating agents: planar compounds which insert into DNA helix and distort backbone (e.g. acridine orange, ethidium bromide)
Physical agents:
UV induced thymine dimers- DNA absorbs UV at 260nm, forms intra-strand pyrimidine dimers, mainly T-T, distortion of double helix ->
prevent replication
Oxygen radicals- cause single and ds breaks -> prevents replication (e.g gamma and x-rays)
Point mutations
Base substitution: transition (purine -> purine, pyrimidine -> pyrimidine) and transversion (pyrimidine to purine)
Base addition or deletion
Greater than one base change mutation
- Addition of deletion of multiple bases
- Inversion of segment of DNA
- Duplication of segment of DNA
- Translocation
Effects of DNA mutation on encoded protein
Synonymous (silent) mutation
Missense mutation (conservative, non-conservative)
Nonsense mutation
Frameshift mutation
Silent mutation
No effect on mutation
Genetic code is redundant. Codes encode same AA
If mutation occurred to swap codons around -> no effect on protein
Missense mutation
One AA in protein is replaced by another
Conservative: replacement with AA of similar biochemical profile -> no loss in protein function
Non-conservative: replacement with AA with different biochemical profile -> complete loss of function or partial loss of function (leaky mutant)
Nonsense mutation
Gives rise to stop codon
TAG, TGA, TAA- stop or nonsense codons which do not encode for an AA, but signal stop translation
Possible effects: limited effect on protein if close to end of open reading frame or cause complete loss of function as premature termination of polypeptide chains -> truncated protein
Frameshift mutation
Caused by nucleotide deletion or insertion of one or more bases -> change in codon reading frame -> change in AA incorporated into protein
-> loss of function but depends on location within gene
Indels not in no. divisible by 3 -> frameshift
Revertant
Strain in which 2nd mutation bas restored phenotype altered by 1st mutation
- True reversion (back mutation): original sequence is restored (ATG -> ATC -> ATG). Point mutations revert, large deletions do not
- Suppression (2nd site mutation): a change at a different site in genome that phenotypically corrects the 1st mutation which is still present
a) intragenic suppression: 2nd mutation is same gene as 1st mutation
b) extragenic (intergenic) suppression: 2nd mutation is a different gene from 1st mutation. Example: mutation in anti-codon of tRNA (2nd mutation) may allow tRNA to recognise nonsense stop codon (1st mutation) and allow protein translation. Mutation -> poor growth
Direct detection mutants
Screening (visual observation or other phenotype)
Disadvantages of screening: very labour-intensive; not all mutations can be visually detected
Indirect detection of mutants: replica plating
Auxotroph has defect in biosynthetic pathway (prototroph is wild type)
Unable to synthesise an essential nutrient
Unable to grow unless supplied with that nutrient
Indirect detection of mutants: selection
Use incubation conditions under which mutant will grow but wild-type will not
Examples pf selectable mutants: antibiotic-resistant mutant, bacteriophage (virus)-resistant, temp-resistant, phototrophic mutant
Direct DNA repair mechanism
Restoration to original undamaged state
Photoreavtivation: photolyase enzyme absorbs energy from 350-500nm light, cleaves dimers back to monomers
Nucleotide excision (short patch) repair
Mismatch repair
Indirect repair
Damage bypass system using DNA replication
Recombination repair: post-replication repair
SOS repair: bypass repair or error-prone repair
Nucleotide excision repair
Products of uvrA, uvrB, uvrC genes form UvrABC exinuclease/UvrABC endonuclease
UvrAB complex migrates up and down DNA until it hits a distortion e.g thymine dimer
UvrA released, UvrC binds and cuts DNA ~4 nt 3’ and 7’nt 5’ of dimer
UvrD helicase helps to remove damaged fragment and UvrBC complex
DNA pol I copies intact strand to fill in gap 3’-5’ using 3’OH primer. Continues repair synthesis for few nt downstream, degrading DNA via its exonuclease activity and simultaneously replacing it
Sealed by DNA ligase
Called ‘short base repair’ as about 13nt replaced
Mismatch repair
During DNA rep, DNA pol III has 3’-5’ exonuclease activity (proof-reading/editing function) -> excises incorrectly base-paired nt at 3’OH end in replicating DNA
Incorrect bases not at 3’end are recognised -> excised by complex containing MutS and MutL (mismatch recognition/excision enzymes) and MutH (strand recognition protein binds methylated DNA)
Complex binds to methylated GATC sequence
Recombinational repair
DNA replication stalls at thymine dimer (DNA pol III adds A, removes A via its 3’-5’ exonuclease editing or proof-reading activity, adds A, removes it etc.)
DNA synthesis reinitiated beyond dimer but gap is lethal to cell
RecA protein binds to ss gap, initiates recombination which fills gap, but leaves gap in parental DNA
Gap filled by DNA pol copying undamaged strand, and DNA ligase
Dimer remains but DNA synthesis can continue, cell survives, has chance to remove dimer vis nucleotide excision repair mechanism
Holliday junctions
Branch migration: holliday junctions move up and down DNA by breakage and rejoining of HB between bases. Can occur spontaneously but is sped up by ATP-hydrolysing RuvAB proteins
How do the 2 DNA molecules separate?
If linear DNA, branch migrates off end
If circular DNA, Holliday structure is resolved by RuvC protein cutting them apart
