Research Starter Grant: Determining the contribution of C to T mutation to the overall mutation rate of a model single-stranded DNA virus

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The successful emergence of many single-stranded DNA (ssDNA) viruses appears to be due to fast evolutionary rates, which must be driven by high mutation rates. However, it is not known how ssDNA viruses could mutate rapidly, given that they replicate by using the high-fidelity DNA polymerases of their host cells. One source of mutation that does not involve polymerase errors is spontaneous chemical degradation of DNA bases. Because ssDNA viruses spend more time single-stranded than single-stranded RNA viruses, their DNA bases are more susceptible to oxidative damage. The most frequent kind of such damage is the deamination of cytosine into uracil, which can lead to mutations of cytosine to thymine when the DNA is replicated. It has already been shown that ssDNA viruses have much higher than expected rates of C to T transitions during their long-term evolution, and this project will investigate whether or not the higher mutation rates of ssDNA viruses is indeed caused by higher C to T mutation rates. The absolute and relative mutation rate of cytosine to the other bases in a model ssDNA virus, bacteriophage phiX174, will be determined. The intellectual merit of this work is its novel cytosine-specific mutation assay, and the combination of phenotypic mutation assays with mutation accumulation studies. This research has the potential for significant broader impacts. An increased understanding of ssDNA viral evolution, and whether or not it is biased towards mutation at cytosines, will allow the design of more complex, but biologically realistic models of mutation that are necessary for accurate molecular epidemiology of emerging ssDNA viruses of plants and animals. As cellular genomes also show evidence of mutation due to chemical degradation (especially in highly transcribed genes, which spend significant time single stranded), these more complex nucleotide substitution models might prove useful in bioinformatic analyses of eukaryotic genes. Most importantly, increased understanding of ssDNA mutational biases could be exploited to combat current and future outbreaks of these emerging pathogens. Additionally, this project contributes to the education and representation of women in science on both graduate and undergraduate levels (in collaboration with the Douglass Project for Women in Science, Math and Engineering).