Comparative Genomics of Chromosome Replication Among Budding Yeasts

忠实的染色体复制是每个分裂细胞的中心任务。尽管DNA复制机制是整个生命树中最保守的机制之一，但启动复制的染色体区域在结构和位置上因物种而有很大差异。染色体复制在模式生物面包师酵母中得到了最好的研究。酵母的复制起始点是由一小段DNA序列定义的，每条染色体上有10到50个起始点。相比之下，人类染色体每条染色体有成百上千个未被DNA序列确定的来源。尽管在起源结构上有这些显著的差异，但人们对复制起源是如何进化的，以及它们可能如何与进化过程中发生的染色体重排有关的知之甚少。贝克酵母和它的其他萌芽酵母亲缘关系在填补我们知识中的这些空白方面提供了无与伦比的优势：许多菌株和物种已经被测序，并适合首先在贝克酵母中进行的实验。这项研究将利用一套染色体范围的分析来发现酵母复制起点，描绘它们的基本序列，并测量每个位置在其天然染色体环境中的调节。此外，将经历过全基因组复制(WGD)的多个酵母物种与其他没有经历过这一事件的物种进行比较的机会，可以为面对大规模基因组剧变时塑造复制和染色体组织的力量提供独特的见解。这项工作将专注于起源如何在5亿年的时间尺度上进化。这些目标建立在检验以下广泛假设的背景下：首先，由于在WGD事件后基因组丢失期间选择的放松，WGD后的物种将比WGD之前的物种表现出更大的起源序列和位置的多样性。第二，相反，近亲物种将共享共同的起源序列，但不会有染色体位置。最后，识别起源的蛋白质机制发生的变化负责推动起源DNA序列的多样化。鉴于起源在维持染色体完整性方面的作用日益受到重视，结果将增加一个重要的缺失元素，以了解染色体结构的进化。广泛的影响：这项工作将在本科生和公共科学教育、纳入代表性不足的群体和科学理解方面产生广泛影响。与海岸线社区学院的新伙伴关系是专门为这一项目提出的，特别是将使更多的学生能够获得研究和以学生为导向、以探究为基础的学习机会。特别是，实习经历将建立信心，并为学生在大学或生物技术研究实验室担任技术人员做好准备，并可能激发他们继续深造的兴趣。所有三名主要调查人员都有良好的本科生教育记录，包括女性、代表性不足的少数族裔，以及主要来自本科院校的学生。开发的方法和分子资源将引起更大的科学界的兴趣，就像这些主要研究人员以前开发的其他技术一样。此外，有助于理解进化过程对科学和社会都是有益的。具体地说，高度保守的过程的快速进化是一个研究不足的现象，结果可能会揭示在其他基本细胞过程中发现的自然变异。

Faithful chromosome replication is a central mission of every dividing cell. Although the machinery for DNA replication is among the most conserved across the tree of life, the regions of chromosomes at which replication initiates 'origins of replication' vary widely across species both in their structure and in their locations. Chromosome replication has been best studied in the model organism baker's yeast. Replication origins in yeast are defined by short stretches of DNA sequence, with ten to fifty origins scattered across each chromosome. In contrast, human chromosomes have many hundreds to thousands of origins per chromosome unspecified by DNA sequences. Despite these striking differences in origin structures, little is known about how origins of replication evolve, and how they may relate to chromosomal rearrangements that take place during evolution. Baker's yeast and its other budding yeast relatives offer an unparalleled advantage in addressing these gaps in our knowledge: many strains and species have been sequenced and are amenable to experiments first performed in baker's yeast. This research will capitalize on the suite of chromosome-wide assays to discover yeast replication origins, delineate their essential sequences, and measure the regulation at each site in its native chromosomal context. Moreover, the opportunity to compare multiple yeast species that had undergone a whole genome duplication (WGD) with other species that hadn't experienced the event can provide unique insights into the forces shaping replication and chromosome organization in the face of a massive genome upheaval. The work will focus on how origins evolved over a 500 million year timescale. The aims are grounded in the context of testing the following broad hypotheses: First, post-­WGD species will show greater diversity of origin sequences and locations than pre-­WGD species due to relaxation of selection during genome loss following the WGD event. Second, in contrast, closely related species will share common origin sequences but not chromosomal locations. Finally, changes that occur to the protein machinery that recognizes origins are responsible for driving the diversification of the DNA sequences of origins. Given the increasingly appreciated role of origins in maintenance of chromosome integrity, the results will add an important missing element to understand the evolution of chromosome structure.Broader Impacts:This work will have broad impacts in undergraduate and public science education, inclusion of underrepresented groups, and scientific understanding. A new partnership with Shoreline Community College has been proposed specifically for this project and will, in particular, result in access to research and student-­driven, inquiry-­based learning for a wider cross-­section of students than would otherwise be possible. In particular, the intern experience will build confidence and prepare students for jobs as technicians in university or biotech research labs, and may spark their interest in pursuing further education. All three principal investigators have strong records of educating undergraduates, including women, underrepresented minorities, and students from primarily undergraduate institutions. The methods and molecular resources developed will be of interest to the larger scientific community, as has been the case for other techniques previously developed by these principal investigators. Furthermore, contributing to the understanding of evolutionary processes is a benefit to science and society. Specifically, rapid evolution of a highly conserved process is an under-­studied phenomenon, and the results are likely to shed light on the natural variation found in other essential cellular processes.