Mutagenesis and Yeast

The Jinks-Robertson Lab at Duke investigates genome stability and mutagenesis using yeast. Saccharomyces cerevisiae is used as a model organism because yeast is easy to grow, and has a cell cycle similar to that of human cells which is also regulated by homologous proteins. The project I am working on deals with mutagenesis in these yeast.

In wild type yeast at the endogenous location of CAN1, it has been found that there is a 2 to 1 strand bias for C to T mutations on the transcribed strand, which are reported as G to A mutations. When DNA mismatch repair is removed by knocking out the MSH6 gene, the strand bias is increased to 9 to 1. Thus, the goal of my project is to try and determine what is driving the mutations bias on the transcribed strand. Over the summer, I will be working with two possible hypotheses: that either 1) the transcribed strand bias is caused by a polymerase that induces mutations during replication or 2) there is a cytosine deaminase that is either more active on the transcribed strand during replication orientation or that there is a preferred deaminase consensus sequence on the transcribed strand.

To test these hypotheses, CAN1 was relocated to ARS306 in both replication orientations. 96 CAN1 mutants will be sequenced from each orientation, and the mutation rates will also be calculated through fluctuations assays. This will also be done in mismatch repair deficient strains, which will be constructed by knocking out MSH6 with KanMX.

Cytosine deamination is the spontaneous loss of an amine group, which converts cytosine to uracil. In humans, the enzyme AID facilitates cytosine deamination, but AID is not present in yeast. This deamination can be repaired, but when not repaired, uracil functions as a normal base, and it is templated by DNA polymerase as Thymine, which creates a C to T mutation. When sequenced, we read this as a G to A mutation. A possible cytosine deaminase that could be causing this transcribed strand bias in yeast is FCY1. Previously this enzyme has been shown to deaminate RNA, but it has not been shown to act on DNA yet. To test this possibility, FCY1 will be knocked out in both orientations using NAT, and the mutants will be isolated using prong plating. 96 of each of these mutants will also be sequenced and the mutation rates will also be calculated through fluctuation assays. This will also be done in FCY1 and mismatch repair deficient strains, which will be constructed by knocking out MSH6 with KanMX.

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Some selection plates for the FCY1 knock outs (a PCR is also run to determine which yeast colonies are FCY1 deficient)

 

 

We’ve already sent out PCR amplified mutants for ARS306 CAN1 strains, and I’m working on prepping the mutants for the yeast with MSH6 knocked out and FCY1 knocked out. The double mutants with both MSH6  and FCY1 are growing, and I still have to do the fluctuation assays. I look forward to continuing this project and getting the results as the summer continues!

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