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

许多单链DNA （ssDNA）病毒的成功出现似乎是由于快速的进化速度，这必须由高突变率驱动。然而，考虑到ssDNA病毒利用宿主细胞的高保真DNA聚合酶进行复制，目前尚不清楚ssDNA病毒是如何快速突变的。一个不涉及聚合酶错误的突变来源是DNA碱基的自发化学降解。由于ssDNA病毒比单链RNA病毒单链时间更长，它们的DNA碱基更容易受到氧化损伤。这种损伤最常见的一种是胞嘧啶脱氨成尿嘧啶，当DNA被复制时，它会导致胞嘧啶突变成胸腺嘧啶。已有研究表明，ssDNA病毒在其长期进化过程中具有远高于预期的C到T的转变率，本项目将研究ssDNA病毒较高的突变率是否确实是由较高的C到T突变率引起的。将测定模型ssDNA病毒噬菌体phiX174中胞嘧啶对其他碱基的绝对突变率和相对突变率。这项工作的智力价值在于其新颖的胞嘧啶特异性突变测定，以及表型突变测定与突变积累研究的结合。这项研究有可能产生更广泛的重大影响。对ssDNA病毒进化的进一步了解，以及它是否偏向于胞嘧啶突变，将允许设计更复杂，但生物学上现实的突变模型，这对于植物和动物中新出现的ssDNA病毒的准确分子流行病学是必要的。由于细胞基因组也显示出由于化学降解而发生突变的证据（特别是在高度转录的基因中，它们在单链上花费大量时间），这些更复杂的核苷酸替代模型可能在真核基因的生物信息学分析中被证明是有用的。最重要的是，增加对ssDNA突变偏差的了解可以用来对抗当前和未来这些新出现的病原体的爆发。此外，该项目还有助于女性在研究生和本科阶段的科学教育和代表性（与道格拉斯科学、数学和工程领域的女性项目合作）。

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).