YEAST TRANSCRIPTION FACTOR INVOLVED IN DNA REPLICATION

我们研究项目的长期目标是深入了解 真核生物DNA复制起始调控机制 使用酵母作为我们的模型系统。 DNA复制的调控 起始是调节细胞生长的一个重要方面。 的 这项建议的目标是根本性的，但可能有有用的 应用于控制致瘤细胞的生长。 在酵母中，被称为自主复制的特定DNA序列 序列（ARS）已被分离。 这些ARS被认为是 染色体上DNA复制的起始位点。 我们研究了 在这些位点上的复制起始机制使用三种不同的 接近。 首先，我们确定了影响ARS功能的基因， 通过分离微染色体维持微染色体 缺陷型（Mcm）突变体。 然后我们克隆并测序了这些基因。 我们 还研究了产物的生化功能和性质， 这些MCM基因。 其次，我们分离出了直接相互作用的蛋白质， 特别是对ARS。 第三，我们分析了 ARS与这些突变体和ARS结合蛋白（ABP）有关。 我们在下一个资助期内的具体目标如下： 将继续研究三种生物的生化和生物功能， MCM基因产物、MCM1基因产物、MCM1、MCM2和MCM3。 特别 将关注MCM1基因产物的分析， 已被证明是一种转录调节因子。 的关系 它在微型染色体维持和转录中的作用 将研究交配型特异性基因的激活。 (2)我们 将进一步确定MCM基因产物在ARS中的作用位点 直接DNA结合研究。 mcm中ARS的缺失分析 突变体已经暗示MCM基因的作用位点 产物位于ARS共有序列的5'侧。 (3)我们将 寻找能与ARS的5'端结合的其它ABP 共有序列

The long term objective of our research project is to gain insight into the regulatory mechanism for DNA replication initiation in eukaryotes using yeast as our model system. The regulation of DNA replication initiation is an important aspect of the regulation of cell growth. The goal of this proposal is fundamental in nature but may have useful applications to the control of the growth of tumorigenic cells. In yeast, specific DNA sequences known as autonomously replicating sequences (ARSs) have been isolated. These ARSs are believed to be the initiation sites for DNA replication on chromosomes. We examined the mechanism of replication initiation at these sites using three different approaches. First, we identified genes that affect the function of ARSs on minichromosomes by the isolation of minichromosome maintenance defective (Mcm) mutants. We then cloned and sequenced these genes. We also examined the biochemical function and properties of the products of these MCM genes. Second, we isolated proteins that interact directly and specifically with ARSs. Third, we analyzed the functional sequence of ARSs with reference to these mutants and the ARS-binding proteins (ABPs). Our specific aims for the next funding period will be as follows: (1) We will continue to study the biochemical and biological functions of three MCM gene products, MCM1 gene products, MCM1, MCM2 and MCM3. Special attention will be given to the analysis of the MCM1 gene product which has been shown to be a transcriptional regulator. The relationship between its role in minichromosome maintenance and transcriptional activation of mating-type specific genes will be investigated. (2) We will further define the sites of action of the MCM gene products at ARSs by direct DNA binding studies. Deletion analysis of ARSs in the mcm mutants has already implicated that the site of action of the MCM gene products is on the 5' side of the ARS consensus sequence. (3) We will look for additional ABPs that would bind to the 5' side of the ARS consensus sequence.