MECHANISM OF DNA-PROTEIN INTERACTION IN DNA REPLICATION

描述：(改编自申请人的摘要)最终目标 本课题旨在了解真核生物dna的作用机制。 复制，特别是滞后链复制，使用酵母， 以酿酒酵母为模型系统。尽管有几个 真核细胞中的DNA聚合酶，其具体作用尚不清楚 很明显，DNA聚合酶α是一种重要的 复制参与启动的聚合酶和Okazaki片段 综合。一种高相对分子质量的多蛋白形式的聚合酶α 从酵母菌及其相关的聚合酶、引物酶、5‘-GT中提纯； 3‘外切酶、单链DNA依赖的ATPase和DNA解旋酶活性 在这个实验室里进行了分析。纯化的5‘-GT的多肽序列分析 3‘端核酸外切酶鉴定为RTH1核酸酶。一种新的DNA 与DNAB解旋酶和SV40T唯一同源的解旋酶 抗原解旋酶已被鉴定。 本项目的具体目标如下：(1)DNA解旋酶A 活性与90、60和50 kDa的三种蛋白共同纯化，并 解旋酶活性位于90 kDa亚基。了解…的作用 解旋酶功能和DNA复制的另外两个亚基， 这些亚基的基因将使用序列信息进行克隆 从纯化的蛋白质中，解旋酶活性将被重组， 并对这些亚基在解旋酶功能中的作用进行了研究。四个 DNA聚合酶α的亚基将被提纯并用于 DNA聚合酶活性的重建及其作用分析 在DNA复制中。 (2)将使用两种杂交技术来鉴定酵母基因 编码的蛋白质与DNA聚合酶α亚单位相互作用，与 酵母DNA解旋酶A和RTH1核酸酶。对于新的基因，基因 研究将探索它们在DNA合成和其他 相关流程。 (3)将进行遗传学研究，以确定酵母DNA的作用 解旋酶A由于解旋酶A对生命力、温度 敏感的条件致命性突变将产生，它们的 用流式细胞术和其他方法评估对DNA复制的影响 技巧。纯化的解旋酶A的生化研究将表征 这种蛋白质的核苷酸酶和解旋酶的活性。

DESCRIPTION: (adapted from applicant's abstract) The ultimate goal of this project is to understand the mechanism of eukaryotic DNA replication, in particular, lagging strand replication, using the yeast, Saccharomyces cerevisiae as a model system. Although there are several DNA polymerases in eukaryotic cells whose specific roles are not clearly defined, it is clear that DNA polymerase alpha is an important replicating polymerase that is involved in priming and Okazaki fragment synthesis. A high molecular weight multiprotein form of pol alpha has been purified from yeast, and its associated polymerase, primase, 5' -> 3' exonuclease, ssDNA dependent ATPase, and DNA helicase activities analyzed in this laboratory. Peptide sequencing of the purified 5' -> 3' exonuclease has identified it to be the RTH1 nuclease. A new DNA helicase which is uniquely homologous to DnaB helicase and SV40 T antigen helicase has been identified. The specific aims of this project are as follows: (1) DNA helicase A activity co-purifies with three proteins of 90, 60, and 50 kDa, and the helicase activity is in the 90 kDa subunit. To understand the roles of the other two subunits in helicase function and DNA replication, the genes for these subunits will be cloned using the sequence information from the purified proteins, the helicase activity will be reconstituted, and the roles of these subunits in helicase function studied. The four subunits of DNA polymerase alpha will be purified and used for reconstituting the DNA polymerase activity and for analyzing their roles in DNA replication. (2) Two hybrid techniques will be employed to identify yeast genes whose encoded proteins interact with subunits of DNA polymerase alpha , with yeast DNA helicase A, and with RTH1 nuclease. For new genes, genetic studies will explore their function in DNA synthesis and in other related processes. (3) Genetic studies will be done to determine the role of yeast DNA helicase A. Since helicase A is essential for viability, temperature sensitive conditional lethal mutations will be generated, and their effects on DNA replication assessed by flow cytometry and other techniques. Biochemical studies of purified helicase A will characterize the nucleotidase and helicase activities of this protein.