A Study of DNA Replication Origins by Comparative Functional Genomics

描述（由申请人提供）：复制起点的活性是调节DNA复制和细胞周期的主要方式。这一过程在芽殖酵母酿酒酵母（Saccharomyces cerevisiae）中得到了最充分的研究，其中起源可以作为质粒上的自主复制序列（ARS）进行研究。然而，在其他生物体中的DNA复制起始机制还没有得到很好的理解。我的初步数据以及其他实验室发表的工作表明，不同的芽殖酵母物种之间的复制起始机制不同。最近丰富的计算工具和测序的基因组允许研究复制起点及其在多个物种中的进化。我建议从不同的酵母物种中分离功能ARS序列，并使用比较基因组学，研究ARS功能的分子决定因素。这将提供深入了解ARS功能的机制以及复制起始机制的演变。 利用成熟的ARS检测结合微阵列分析，我将从不同种类的芽殖酵母中分离和定位ARS。我将使用比较基因组方法来表征不同酵母所利用的ARS识别机制。我还将分析ARS元件的进化及其基因组位置。为了确定ARS识别蛋白的差异，负责ARS功能的差异，我将进行一系列物种间ARS和蛋白质交换实验。相关性：DNA复制的启动是基因组动力学和细胞周期调控的核心参与者。因此，这一过程中的缺陷与基因组不稳定和癌症有关也就不足为奇了。这些研究将有助于我们通过追踪ARS和ARS识别蛋白在2亿年进化时间内的共同进化来理解复制起始的分子机制。此外，我们还将了解来自不同酵母的蛋白质如何与不同的ARS序列相互作用。从长远来看，这些信息将有助于了解DNA复制如何在人类细胞中启动，以及这个过程中的缺陷如何导致癌症。

DESCRIPTION (provided by applicant): The activity of replication origins is a major way of regulating DNA replication and the cell cycle. This process has been most well studied in the budding yeast Saccharomyces cerevisiae, where origins can be studied as autonomously replicating sequences (ARSs) on plasmids. However, DNA replication initiation mechanisms in other organisms are not well understeood. My preliminary data as well as published work from other labs suggests that replication initiation mechanisms differ among different budding yeast species. The recent abundance of computational tools and sequenced genomes allows the study of replication origins and their evolution across multiple species. I propose to isolate functional ARS sequences from different yeast species and, using comparative genomics, study the molecular determinants of ARS function. This will provide insight into both the mechanisms of ARS function as well as the evolution of replication initiation mechanisms. Using the well developed ARS assay combined with microarray analysis, I will isolate and map ARSs from different species of budding yeast. I will use comparative genomic approaches to characterize the mechanisms of ARS recognition utilized by the different yeasts. I will also assay the evolution of ARS elements and their genomic locations. To identify the differences in ARS recognition proteins that are responsible for differences in ARS function, I will perform a series of inter-species ARS and protein swapping experiments. Relevance: The initiation of DNA replication is a central player in genome dynamics and cell cycle regulation. It is therefore not surprising that defects in this process have been linked to genomic instability and cancer. The proposed studies will contribute to our understanding ofthe molecular mechanisms of replication initiation by tracking the co-evolution of ARSs and ARS recognition proteins across 200 million years of evolutionary time. In addition, we will learn how proteins from different yeasts interact with different ARS sequences. In the long-term, this information will be useful in understanding how DNA replication initiates in human cells and how defects in this process can contribute to cancer.