LECTURE 2 (The molecular basis of mutation 2) Flashcards
What are spontaneous mutations? what’s their frequency?
they arise in the absence of a mutagen and have a frequency of 10^-4 in retrovirus, 10^-9 in euk
Are spontaneous mutations due to external stimuli or due to a pop?
Fluctuation test: this was addressed by luria & delburk, they won a nobel price. E.coli were plated in the presence of phage T1 (infects and kills the bact but some bact can survive) they let 20 independent cultures to grow for a few days until they were 20^8 and then analyze them and also 10 aliquots from a large culture also let them grow until they were 10^8 . the result show that in the independent cultures the numbers of T1 mutants vary so much whereas in the large cultures all the aliquots had more or less the same amount of T1 mutants, this means that the mutations were not due to the T! phage otherwise all the cultures would have the same amount of mutants because all were exposed to the same amount of phages.
How do we get the mirror image bt plates in replica plating?
by using a velvet covered stamp
Can the existence of mutants in a population before selection be demonstrated directly?
yes through replica plating: Lederberg
we plate colonies in a master plate (non-selective medium) therefore no phage and all colonies grow. Then we imprint with a velvet stamp and stamp on different plates w/ phage (same amount), we can compare the plates bc they’re mirror images and we realize that some colonies in the 1st rich medium were resistant and all the replica plates show the same pattern. This suggests that mutations occurred before exposure to T1 phage otherwise different patterns would be observed.
This can be used to screen auxotrophic mutants.
How can spontaneous mutations occur?
By errors in DNA replication or spontaneous lesions
What are the most common spontaneous lesions?
- deamination and depurination and oxidative damage
What is deamination? give examples
It’s a way of causing an spontaneous lesion. loss of an amine.
e. g deamination of cytosine leads to uracil so instead of a GC pair we get a AU pair (AT at the level of DNA)
e. g deamination of 5’methylcytosine generates thymine (5’methyluracil) again GC to AT pairing.
How can a deamination be observed at the level of DNA?
because if there’s a U it shouldn’t be there. so uracil glycosilates initiate the base excision repair mechanism
What is depurination?
The loss of a purine this occurs quite often in mammals 10000npurines are lost every 20h and this is quite critical. sometimes another base is inserted in the apurinic site but if it’s the wrong one then it’s a mutation
What are oxidative damaged lesions?
this occurs due to oxygen e.g the 8-oxodG produced due to radiation (G pairing with A) these oxidative lesions cause many humans diseases.
What are errors in DNA duplication?
- base substitutions in already present bases or new bases. tautomeric shift can cause misfiring in DNA replication.
- indels (leading to frameshift)
How are indels caused in DNA replication?
via a mechanism called replication slippage. indels arise when loops in ss regions are stabilized by slipped mispairing of repeated seq
Give an example of replication slippage in nature.
in the LacI gene of E.coli there are CTGG sequences repeated mutations can occur here if there’s an addition or loss of CTGG sequences. if there’s an addition DNAP needs to complete that addition plus the rest of the molecule therefore you end up with a much larger seq. the majority of the mutations in this lacI gene result from the addition of ctgg sites rather than the deletion
Appart from replication slippage, can indels appear through another mechanism?
yes, they can appear due to offset homologous recombination between copies of the repeats.
What are the main DNA repair mechanisms?
- direct reversal of damage
- base excision repair (BER)
- nucleotide excision repair
- mismatch repair
- non-homologous end joining
- homologous recombination
