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复制启动的调控机制 使用酵母作为我们的模型系统。DNA复制的调控 启动是细胞生长调控的一个重要方面。这个 该提案的目标本质上是基本的，但可能会有帮助 在控制致瘤细胞生长方面的应用。 在酵母中，特定的DNA序列被称为自主复制 序列(ARs)已被分离。这些ARS被认为是 染色体上DNA复制的起始点。我们检查了 在这些位置使用三种不同的复制启动机制 接近了。首先，我们确定了影响ARSS功能的基因 从微染色体维护的分离谈微染色体 缺陷(MCM)突变体。然后，我们对这些基因进行了克隆和测序。我们 还检测了产品的生化功能和性质 这些MCM基因。第二，我们分离了直接相互作用的蛋白质 特别是在抗逆转录病毒药物方面。第三，我们分析了该基因的功能序列 ARSS涉及这些突变体和ARS结合蛋白(ABPs)。 我们下一个资金阶段的具体目标如下：(1)我们 将继续研究三种生物化学和生物功能 MCM基因产物、MCM1基因产物、MCM1、MCM2和MCM3。特价 将注意对MCM1基因产物的分析 已被证明是一种转录调节因子。两国关系 它在微染色体维持和转录中的作用 交配型特定基因的激活将被研究。(2)我们 将进一步确定MCM基因产物在ARSS中的作用部位 通过直接的DNA结合研究。MCM中ARSS的缺失分析 突变体已经暗示了MCM基因的作用部位 产品位于ARS共识序列的5‘端。(3)我们会 寻找将与ARS的5‘侧结合的额外的ABP 共识序列。

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.