Molecular Genetic and Genomic Analysis of Mutation Clusters Induced by Ubiquitous Endogenous Mutagenic Processes in Highly Sensitized Yeast Model Systems

在35亿年的时间里，地球上进化出了惊人的生物多样性。自然选择的进化作用于生命形式种群中的遗传变异，因此具有有利遗传特征的个体更有可能生存下来并将其基因传递给下一代。这种遗传变异大多以突变的形式发生，即DNA信息编码内容的改变。突变总是以非常低的“背景”频率发生，但这些突变是如此罕见，以至于它们的起源很难研究，也不能很好地理解。******细胞内有许多分子可能会破坏DNA，导致突变。我们将研究两个相互关联的过程的作用，这两个过程可以产生损伤DNA的反应性分子：氧代谢和食物利用。我们高度敏感的面包师酵母模型系统产生长长的单链DNA，它比细胞中常见的双链DNA更容易发生突变。事实上，对暴露的单链DNA的损害通常会产生密集间隔的突变簇。这对于生成大量突变是理想的，我们将对其进行计算分析以推断特征突变签名。突变标记是由于1)优先靶向（或避免）特定DNA序列上下文和2)DNA碱基序列中不同的替换模式而产生的分子指纹，这些替换模式可以是C到A、C到G、C到T、T到A、T到C或T到G的组合。我们将利用酵母系统全面表征不同条件下氧代谢和食物利用的突变标记。然后，我们将检查各种数据库，寻找这些相同的特征，并推断这些过程在进化过程中塑造人类和许多其他物种的基因组中的作用。******进化理论预设存在遗传变异以供自然选择之用，但没有说明遗传变异最初从何而来。我们的计划旨在回答长期存在的，使生命进化的遗传变异起源的基本问题。除了对地球生物多样性的最终基础产生重要的见解外，还有潜在的应用，用于具有理想特性的微生物的简易工程，包括生物燃料或制药生产；生物修复;或者合成生物学。利用突变簇，我们可以很容易地在活微生物中创建一个特定目标基因的许多变体，而不会在整个微生物基因组中引入许多其他突变。因此，我们项目的成功也可以加速其他领域的进展。该计划将加强加拿大在有影响力的纯科学和应用科学研究方面的领导地位。

An amazing diversity of life has evolved on Earth over some 3.5 billion years. Evolution by natural selection acts on the genetic variation within a population of life forms, such that individuals with advantageous genetic traits are more likely to survive and pass on their genes to the next generation. Much of this genetic variation occurs in the form of mutations, which are changes to the information coding content of DNA. Mutations occur all the time at a very low "background" frequency, but these mutations are so rare that their origins are difficult to study and not well understood.******There are many molecules within cells that potentially can damage DNA, leading to mutations. We will investigate the roles of two interrelated processes that can create reactive molecules which can damage DNA: oxygen metabolism and foodstuff utilization. Our highly sensitive baker's yeast model system generates long stretches of single stranded DNA, which is about 100 to 1,000 times more susceptible to being mutated than the usual double stranded DNA found in cells. In fact, damage to the exposed single stranded DNA often yields clusters of closely spaced mutations. This is ideal for generating large numbers of mutations, which we will analyze computationally to infer the characteristic mutation signatures. A mutation signature is a molecular fingerprint due to 1) preferential targeting (or avoidance) of specific DNA sequence contexts and 2) distinctive patterns of substitutions in the DNA base sequence, which can be some combination of C to A, C to G, C to T, T to A, T to C, or T to G. We will comprehensively characterize the mutation signatures of oxygen metabolism and foodstuff utilization under different conditions using the yeast system. We will then check various databases to look for these same signatures and infer the role of these processes in shaping the genomes of humans and many other species over the course of evolution.******The theory of evolution presupposes that there is genetic variation for natural selection to act upon, but says nothing about where the genetic variation comes from in the first place. Our program seeks to answer the longstanding, fundamental question of the origins of genetic variation that enables life to evolve. In addition to yielding important insights into the ultimate basis for biodiversity on Earth, there are potential applications for facile engineering of microbes with desirable properties, including for biofuel or pharmaceutical production; bioremediation; or synthetic biology. Using mutation clusters, we can easily create many variants of a specific target gene of interest in living microbes, without the drawback of introducing many other mutations throughout the microbes' genomes. Success in our program can therefore accelerate progress in these other fields as well. This program will bolster Canada's status as a leader in impactful pure and applied scientific research.