Initiation of Eukaryotic DNA Replication

描述（由申请人提供）： S期的DNA复制对维持基因组的完整性至关重要， 真核细胞因此，了解其发生机制和调控规律， 复制起始对人类健康至关重要。在过去 二十年来，酵母的遗传方法已经确定了特定的DNA序列， 激活复制起始，以及几个起始因子 (ORC、cdc 6、MCM、cdc 45），其在所有真核生物中是保守的。但在许多 这些因子的分子功能及其调控 仍然知之甚少。此外，不太可能所有因素 已确定复制启动所需的参数。解决这些 问题，一个通用的无细胞系统是必不可少的。使用非洲爪蟾卵 提取物，该研究人员最近开发了第一个体外系统， 支持真核DNA复制的起始， 蛋白质环境该系统支持-100%高效的DNA 细胞周期调控的复制。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， 真核生物的起源。在具体目标1中，新型染色质结合试验 将被用来开发一个简单的模型的空间安排， 复制前复合体中的起始因子。具体目标2 cdk 2/cyclin E蛋白激酶催化cdc 45结合的机制 将对复制起点进行研究。在具体目标3中， 将确定复制起点处的DNA何时首先变性。在 具体目标4，解旋起始的DNA解旋酶将被表征。 在特定目标5中，我们研究了染色质的一种新模式的机制， 通过MCM复合物结合。预计，拟议的研究将 对真核生物复制起始和 提供了一个坚实的基础，以调查这一过程是如何调节，以及如何 它可能在人类疾病中被错误调节。

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.