Initiation of Eukaryotic DNA Replication

描述(由申请人提供)：对启动的准确规定 S期的DNA复制是维持猪基因组完整性的关键 真核细胞。因此，理解这一现象的机制和调控 复制启动对人类健康至关重要。在最后一次 二十年来，酵母中的遗传方法已经确定了特定的DNA序列 激活复制启动，以及几个启动因子 (ORC，cdc6，mcm，cdc45)，在所有真核生物中都是保守的。然而，在许多情况下 这些因子的分子功能及其调控 人们对此仍然知之甚少。此外，所有因素都不太可能 已经确定了启动复制所需的基因。要解决这些问题 问题是，一个多功能的无细胞系统是必不可少的。利用非洲爪哇的卵 提取物，这位研究人员最近开发了第一个体外系统 支持在完全可溶的情况下启动真核DNA复制 蛋白质环境。该系统支持-100%高效的DNA 受细胞周期调控的复制。因此，它代表着一种独特的 有机会对复制进行详细的生化分析 真核生物中的发端。在特定目标1中，新的染色质结合分析 将被用来开发一个简单的模型，用于 复制前复合体中的启动因素。在具体目标2中， CDK2/Cyclin E蛋白激酶催化cdc45结合的机制 将对复制的起源进行调查。在具体目标3中，时间 复制起始处的DNA何时第一次变性将被确定。在……里面 具体目标4，将描述解开起始点的DNA解旋酶。 在特定的目标5中，我们研究了一种新的染色质模式的机制 被MCM复合体结合。总之，拟议的研究预计将 极大地促进了对真核细胞复制启动和 提供坚实的基础，以调查此过程是如何监管的以及如何 在人类疾病期间，它可能被错误地调节。

DESCRIPTION (provided by applicant): Precise regulation of the initiation of DNA replication during S phase is essential to maintain genome integrity in eukaryotic cells. Therefore, understanding the mechanism and regulation of replication initiation is of critical importance for human health. In the last two decades, genetic approaches in yeast have identified specific DNA sequences that activate replication initiation, as well as several initiation factors (ORC, cdc6, MCM, cdc45) that are conserved in all eukaryotes. However, in many cases, the molecular functions of these factors as well as their regulation remain poorly understood. In addition, it is unlikely that all the factors required for replication initiation have been identified. To address these issues, a versatile cell-free system is essential. Using Xenopus laevis egg extracts, this investigator recently developed the first in vitro system that supports initiation of eukaryotic DNA replication in a completely soluble protein environment. This system supports -100 percent efficient DNA replication that is cell-cycle regulated. As such, it represents a unique opportunity to carry out a detailed biochemical analysis of replication initiation in eukaryotes. In Specific Aim 1, novel chromatin-binding assays will be used to develop a simple model for the spatial arrangement of initiation factors in the pre-replication complex. In Specific Aim 2, the mechanism by which cdk2/cyclin E protein kinase catalyzes association of cdc45 with origins of replication will be investigated. In Specific Aim 3, the time when DNA at the origin of replication is first denatured will be determined. In Specific Aim 4, the DNA helicase that unwinds the origin will be characterized. In Specific Aim 5, we examine the mechanism of a novel mode of chromatin binding by the MCM complex. Together, the proposed studies are expected to significantly advance understanding of eukaryotic replication initiation and provide a firm foundation to investigate how this process is regulated and how it might be mis-regulated during human disease.