EUKARYOTIC DNA REPLICATION

在阐明DNA的作用机制方面已取得了相当大的进展 在原核生物和真核生物中的复制。这些研究强调， 其复杂性和基本相似性。即使有这样的进步，我们 还远未了解其复杂性，我们最近才开始 了解它的调节以及与DNA修复和细胞 增长DNA复制一直是抗癌治疗的目标， 多年来对这些特工的工作方式知之甚少因此，如果我们 要理解异常增长，我们必须了解 DNA复制发生。 在上一个资助期内，我们研究了 猿猴病毒（SV）40 DNA复制的机制。我们有 用纯化的蛋白质重建复制途径。我们有 研究了SV40 T抗原，唯一需要的病毒蛋白， 激活SV4O起源，并通过其内在的DNA解旋酶 参与复制反应。识别、隔离 和其他蛋白质的作用机制，由宿主提供， 产生了关于它们在染色体复制中的作用的重要信息。 在这项赠款的有效期内，我们将研究 许多宿主蛋白质参与SV 4 O DNA复制。这些 包括l）组装所需的辅助蛋白的机制， 赋予催化聚合酶高的持续合成能力。夹具装载机， activator 1（A1，也称为RFC）包含五个不同的子单元， 被克隆了。我们将研究每个亚基在 增殖细胞核抗原的载量 它作为一个滑动夹，将聚合酶系在DNA上， DNA.我们将研究A1分子沿着DNA追踪的机制， 它找到引物末端。2)PCNA的作用将通过特定的 强调各种生长调节剂（p21，Gadd45）的影响， 与PCNA相互作用并影响其作用。3)的合成 三聚体DNA结合蛋白，称为人单链结合蛋白 (or RPA）将进行调查。HSSB是唯一的结合蛋白， 支持SV40复制。所有的亚单位都被克隆了， 表达。我们将研究它的合成和每个亚基的作用， SV4O途径。3)我们计划研究 粟酒裂殖酵母DNA原始序列的可用性， 作为质粒中的自主复制序列（ARS），与 识别相互作用的起始复制复合体（ORC）， 酿酒酵母的起源引起了我们的注意， 这系统.我们计划从S.粟酒和 进一步表征了从S.蓬贝， 与S.粟酒裂殖酵母保守的核心序列。

Considerable progress has been made in elucidating the mechanism of DNA replication in prokaryotes and eukaryotes. These studies have emphasized its complexity and fundamental similarities. Even with this progress, we are far from understanding its intricacies and we have only recently begun to appreciate its regulation and relationship to DNA repair and cell growth. DNA replication has been the target of anticancer therapies for years with little appreciation of how these agents work. Thus, if we are to understand aberrant growth, we must have fundamental knowledge of how DNA replication occurs. During the previous grant period, we have studied the biochemical mechanism by which simian virus (SV) 40 DNA is replicated. We have reconstituted the replication pathway with purified proteins. We have investigated how SV4O T antigen, the only viral protein required, activates the SV4O origin, and through its intrinsic DNA helicase participates in the replication reaction. The identification, isolation and mechanism of action of the other proteins, supplied by the host, have yielded important information about their role in chromosomal replication. During the tenure of this grant, we will investigate the mode of action of a number of the host proteins involved in SV4O DNA replication. These include l) the mechanism of assembly of the accessory proteins needed to confer high processivity onto the catalytic polymerases. The clamp loader, activator 1 (A1, also called RFC), contains five different subunits all of which have been cloned. We will investigate the role of each subunit in the loading of the processivity factor proliferating cell nuclear antigen (PCNA) onto DNA which acts as a sliding clamp tethering the polymerase to DNA. We will investigate the mechanism by which A1 tracks along DNA until it finds primer ends. 2) The role of PCNA will be examined with particular emphasis on the influence of various growth regulators (p21, Gadd45) which interact with PCNA and influence its action. 3) The synthesis of the trimeric DNA binding protein, called human single stranded binding protein (or RPA) will be investigated. HSSB is the only binding protein that supports SV40 replication. All of the subunits have been cloned and expressed. We will examine its synthesis and the role of each subunit in the SV4O pathway. 3) We plan to investigate the replication of Schizosaccharomyces pombe DNA. The availability of an origin sequence that acts as an autonomous replication sequence (ARS) in plasmids, coupled with the identification of the origin replication complex (ORC) which interacts with the origin of Saccharomyces cerevisiae has focused our attention to this system. We plan to isolate the ORC polypeptides from S. pombe and to further characterize two distinct protein isolated from S. pombe that interact with the S. pombe conserved core sequence.