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，而这必然是由高突变率驱动的。然而，鉴于 ssDNA 病毒通过使用宿主细胞的高保真 DNA 聚合酶进行复制，目前尚不清楚它们如何快速突变。不涉及聚合酶错误的突变来源之一是 DNA 碱基的自发化学降解。由于 ssDNA 病毒比单链 RNA 病毒处于单链状态的时间更长，因此它们的 DNA 碱基更容易受到氧化损伤。 最常见的此类损伤是将胞嘧啶脱氨基为尿嘧啶，这会导致 DNA 复制时胞嘧啶突变为胸腺嘧啶。 已经表明，单链DNA病毒在其长期进化过程中的C到T转变率远高于预期，本项目将研究单链DNA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。 将确定模型 ssDNA 病毒（噬菌体 phiX174）中胞嘧啶对其他碱基的绝对和相对突变率。 这项工作的智力优势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影响。对单链DNA病毒进化以及它是否偏向于胞嘧啶突变的更多了解，将允许设计更复杂但生物学上真实的突变模型，这对于新兴植物和动物单链DNA病毒的准确分子流行病学是必需的。 由于细胞基因组还显示出由于化学降解而导致突变的证据（特别是在高度转录的基因中，这些基因花费大量时间处于单链状态），因此这些更复杂的核苷酸替换模型可能在真核基因的生物信息学分析中有用。最重要的是，加深对 ssDNA 突变偏差的了解可以用来对抗这些新兴病原体当前和未来的爆发。此外，该项目还致力于促进研究生和本科生科学领域女性的教育和代表性（与道格拉斯科学、数学和工程领域女性项目合作）。

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).