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).
While the same evolutionary principles apply to everything on earth – viruses, bacteria, plants and humans – not all organisms evolve in the same way. Viruses, in particular, have many different genome types and these distinct genomes could lead to different molecular evolutionary patterns. For instance, long-term evolutionary studies of single-stranded DNA viruses indicate that cytosines are less frequent in their genomes than the other nucleotides, and suggest that the missing cytosines have been replaced by thymines. We studied the mutation rate and evolution of the single-stranded DNA virus phiX174, which infects bacteria. We asked whether cytosine mutates at a higher rate than the other bases, which could help explain the long-term evolutionary patterns. Our experiments involved generating mutants in the start codon that replaced a thymine with a cytosine, which we could use to measure the rate of reversal back to thymine. Our ongoing work addresses this with both exact measures of the cytosine mutation rate and comparing the accumulated mutations in portions of the phiX174 genome. This work gives a mechanistic justification for modeling some kinds of viral evolution differently than we model the evolution of cellular organisms, which could impact viral epidemiology during outbreaks of diseases. This research also contributed to the education and career development of a graduate student and an undergraduate student.
