18 - DNA mutation and repair* Flashcards
Gene
DNA sequence encoding for a polypeptide, tRNA or rRNA
Genotype*
Collection of genes an organism has (genetic composition)
Phenotype*
Observable characteristics of an organism (expression of genes). One phenotype has multiple possible genotypes
Mutation
A permanent, heritable change to the base sequence of DNA
Wild type
Organism as it was first isolated from nature (normal type)
Mutant
Organism that differs from wild type as a result of mutation
How often do mutations occur
1 in 10^7 to 10^11 base pairs
3 types of mutagens
- DNA modifying agents
- Intercalating agents
- Physical agents (Thymine dimers, oxygen radicals)
DNA modifying agents
Add alkyl groups to bases and change base pairing. e.g. ethylmethane sulphonate (EMS) adds ethyl groups
Intercalating agents
Planar compounds which insert into DNA helix and distort the backbone (e.g. acridine orange, ethidium bromide)
UV Thymine dimers
UV induced Intra-strand pyrimidine dimers (mainly T-T) that distort double helix and prevent replication
Oxygen radicals
Cause single and double stranded breaks, prevents replication (e.g. gamma and x rays)
Point mutations
- Base substitution (transversion and transition)
- Base addition or deletion (indel)
Transition base substitution
Purine replaced with purine or pyrimidine replaced with pyrimidine
Transversion base substitution
Purine replaces pyrimidine or pyrimidine replaces purine
Greater than one base change mutations
- Addition or 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 and non-conservative)
- Nonsense mutation
- Frameshift mutation
Silent mutation
No effect on protein (or amino acid sequence)
Missense
amino acid replaced by different amino acid
Conservative missense
Replacement with an amino acid of similar biochemical profile. No loss in protein function
Non-conservative missense
Replacement with an amino acid of different biochemical profile. Complete or partial loss of function (leaky mutant)
Nonsense mutation
gives rise to stop codon (UAA, UGA, UAG). Limited effect (if close to end of reading frame) or complete loss of function (truncated protein)
Frameshift
Caused by nucleotide deletion of insertion which changes reading frame
Revertant
a strain in which a 2nd mutation has restored the phenotype altered by a 1st mutation
True reversion (back mutation)
Original sequence is restored. Point mutations revert, large deletions do not.
Suppression (2nd site mutation)
A change at a different site in the genome that phenotypically corrects the 1st mutation which is still present. (intragenic and intergenic/extragenic)
Intragenic suppression
second mutation is in same gene as first
Extragenic/intergenic suppression
second mutation is in different gene from first
Direct detection of mutants
Screening (visual observation)
Disadvantages of screening
Labour intensive and not all mutations can be visually detected
Indirect detection of mutants
Replica plating and selection
Replica painting example
- Auxotroph has defect in a biosynthetic pathway (prototroph is wild type)
- Unable to synthesise an essential nutrient
- Unable to grow unless supplied with that nutrient
- Two plates compared, one with and one without nutrient
Selection
use incubation conditions under which the mutant will grow but the wild-type will not (e.g. antibiotic resistance)
other examples of selectable mutants
- bacteriophage resistant mutant
- temperature resistant mutant
- prototrophic mutant
Direct repair
Restoration to original undamaged state
Examples of direct repair
- Photoreactivation
- Nucleotide excision repair (short patch repair)
- Mismatch repair
Indirect repair
Damage bypass system using DNA replication (e.g. recombination repair)
Nucleotide excision repair
- Products of uvrA, uvrB and uvrC genes form 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
- UvrD helicase removes damaged fragment and releases UvrBC complex
- DNA polymerase 1 fills in gap and DNA ligase joins fragments
- ~13nt replaced
Mismatch repair at 3’ end of replicating DNA
DNA polymerase 3 has 3’-5’ exonuclease activity (proof reading/editing function)
Mismatch repair not at 3’ end
- MutS protein slides along DNA until mismatch is found.
- MutL binds MutS and that complex binds to MutH, which is already bound to hemimethylated sequence
- MutH makes cut in nonmethylated strand and base mismatch is digested
- DNA polymerase fills in and ligase seals ends
Recombinational repair
- DNA replication stalls at thymine dimer and gap is produced which is lethal to cell
- RecA protein binds to ss gap and initiates recombination which fills gap by removing the correct homologous DNA from the donor DNA leaving a new gap
- Gap filled by DNA polymerase copying
undamaged strand, and DNA ligase - Dimer remains but cell survives and can use NER
Holliday junctions
branched nucleic acid structure that contains four double-stranded arms joined. Occurs during homologous recombination
Branch migration of holliday junctions
Holliday junctions move up and down the DNA by breakage and rejoining of H bonds between bases. Can happen spontaneously but is sped up by ATP hydrolysing RuvAB proteins
How do two DNA molecules separate in a holliday junction
- Linear DNA: branch migrates off the end
- Circular DNA: RuvC protein cuts them apart
