Initiation of Eukaryotic DNA Replication

描述（由申请人提供）： 启动的精确监管 S期的DNA复制对于维持基因组完整性至关重要 真核细胞。因此，了解其机制和调节 复制启动对于人类健康至关重要。在最后 二十年来，酵母的遗传方法已经鉴定出特定的 DNA 序列 激活复制起始以及几个起始因子 （ORC、cdc6、MCM、cdc45）在所有真核生物中都是保守的。然而，在许多 这些因素的分子功能及其调控 仍然知之甚少。此外，所有因素都不太可能 复制启动所需的已确定。为了解决这些 问题，多功能的无细胞系统至关重要。使用非洲爪蟾卵 提取物，这位研究人员最近开发了第一个体外系统 支持在完全可溶的环境中启动真核 DNA 复制 蛋白质环境。该系统支持-100% 高效 DNA 复制受细胞周期调节。因此，它代表了一种独特的 有机会对复制进行详细的生化分析 真核生物中的起始。在具体目标 1 中，新型染色质结合测定 将用于开发一个简单的空间排列模型 复制前复合物中的起始因子。在具体目标 2 中， cdk2/cyclin E蛋白激酶催化cdc45结合的机制 将研究复制起点。在具体目标 3 中，时间 复制起点的DNA何时首次变性将被确定。在 具体目标 4，解旋起始点的 DNA 解旋酶将被表征。 在具体目标 5 中，我们研究了染色质新模式的机制 通过 MCM 复合物结合。总之，拟议的研究预计将 显着促进对真核复制起始和 为研究如何监管这一过程以及如何 在人类疾病期间它可能会被错误调节。

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.