Mutation and repair Flashcards
what is a point mutation?
mutation that involved a change in a single base
what is a missense mutation
a point mutation that causes a codon to change into a different codon
what is a nonsense mutation?
a point mutation that causes a codon to change into a stop codon
what is an indel mutation
insertion of deletion of base(s), cause frameshift
what is frameshift
when a base(s) is inserted or deleted, the subsequence nucleotides are all shifted up by one, causing all the codons to be out of phase
what are mutagens
external factors that cause mutations e.g.:
- viruses
- UV
- chemicals
what can cause error during translation
for example:
- mispairing of amino acid to tRNA by aaRS (aminoacyl tRNA synthetase)
- misparing of codon to anticodon by ribosome
DNA is prone to spontaneous & externally-induced lesions, what are leisons?
- damage/error in a base in dsDNA
- still can be corrected because complimentary base pair stores original information
- if not correct will lead to mutation after replication (original information lost)
what are some of the reactions that cause leisons?
- Oxidative deamination
- Depurination
- Thymine dimerisation
describe oxidative deamination
- C change to U
- by free radicals
describe depurination
- A (or G) loses its base, resulting in a hole
- glycosidic bond between base and ribose broken
- by spontaneous hydrolysis
describe thymine dimerisation
2 adjacent thymines dimerise to form a cyclobutane dimer
5-bromouracil (5BrU) is a potent mutagen, how?
- Unlike ‘normal’ bases, 5BrU equilibrium mix is even-ish, rather than heavily biased to keto form
what does keto-5BrU pair to
A
what does enol-5BrU pair to
G
How does 5BrU cause mutation?
- first, for example there is a T:A base pair
- replication occurs, keto-5BrU as a free nucleotide comes in and pair with A
- next round of replication, keto-5BrU has tautomerised and become enol-5BrU, so pairs with incoming G
- another round of replication, G pairs with C
- results in G:C base pair, original information (T:A) lost
how can small leisions where a base is altered in DNA be repaired?
- base excision
- DNA glycocylase recognises altered base (different DNA glycocylases recognise different kinds of damage, e.g. uracil DNA glycocylase checks for U, which is a deaminated C)
- DNA glycocylase hydrolyses glycosidic bond between base and sugar, removing the base
- AP endonuclease and phosphodiesterase cuts out the sugar phosphate with a missing base
- DNAP then synthesise DNA to repair the hole
- ligase seals sugar phosphate backbone
what is AP endonuclease
an enzyme that recognizes and cuts out any site in the DNA helix that contains a deoxyribose sugar with a missing base
How does the cell know which base is incorrect?
- cell can know which strand is parental
- DNA repair has to happen at the same time as DNA replication or else original information is lost
how does the distinction between daughter strand and parental strand occur?
in lagging strand:
- obvious because lagging strand has multiple okazaki fragments and primers
in leading strand:
- parental strand has more methylation due to age, this can be detected and allow recognised
how can large leisions be repaired?
- nucleotide excision
- homologous recombination
how does nucleotide excision occur?
bacteria:
- multienzyme complex recognise a lesion (e.g. pyrimidine dimer), one cut is made on each side of the lesion
- DNA helicase then removes the entire portion of the damaged strand, leaves the gap of 12 nucleotides
humans:
- damaged DNA is recognized
- helicase unwinds the DNA duplex locally
- excision nuclease cleaves on either side of the damage, leaving a gap of about 30 nucleotides
both humans and bactera:
- DNAP repairs gap, ligase seals backbone
what is a truncated mRNA
- an mRNA which transcription has not been completed => does not have stop codon
- RNAP is error prone, sometimes stops aruptly, mRNA without a stop codon
what would a truncated mRNA cause?
- truncated mRNA does not have stop codon
- release factors recognise stop codon, without stop codon, mRNA cannot be released from ribosome
- ribosome is stalled, rendered useless
what do prokaryotes use to deal with truncated mRNA?
- tmRNA = transfer messenger mRNA
- a tRNA and mRNA hybrid
- tRNA domain can bind to peptidyl transferase active sites
- mRNA domain of tmRNA can bind to SSU where truncated mRNA would be
- mRNA domain of tmRNA encodes degradation tag
(prokaryotes) How does tmRNA deal with truncated mRNA
- stalled ribosome recruited by EF-Tu
-
EF-Tu:
- shuffles tRNA domain of tmRNA to A site, and mRNA domain of tmRNA is pushed into SSU where mRNA is
- truncated mRNA removed
- hydrolyse one GTP
-
EF-G:
- continues translation so that the petidyl group translated from truncated mRNA is transfered onto tmRNA
- the codons on mRNA domain of tmRNA is translated until stop codon is reached and tmRNA is then released
- specific sequence in mRNA domain of tmRNA taggs polypeptide produced for degradation by protease
which two points must fidelity be maintained during translation?
- ribosome decoding: codon:anticodon
- tRNA aminoacylation: tRNA: amino acid
how does aaRS (aminoacyl tRNA synthetase) maintain fidelity during tRNA aminoacylation?
- chances of aaRS picking up the wrong tRNA is small because tRNA is large, can be readily recognised by many of its features (e.g. anticodon)
- chances of aaRS picking up the wrong amino acid is high because amino acids are small and can be similar (e.g. valine and isoleucine)
- aaRS has esterase proofreading site to deal with this problem, reducing error to 10-5 per amino acid
describe how the esterase proofreading site may help IleRS (isoleucine tRNA synethetase) to not accidentally add valine to tRNA
- esterase proofreading site is smaller than aminoacylation site, can only accomodate valine
- if valine is accidentally added, esterase proofreading site (which only valine can fit in) will remove valine
- because esterase proofreading site can only accomodate valine, isoleucine will not be removed by it
