BIOCHEMICAL STUDIES OF HUMAN DNA POLYMERASE DELTA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 拟议的研究代表了我们长期目标的继续，即了解人类DNA聚合酶Delta的酶学和功能，该酶是哺乳动物染色体DNA复制的中心酶。该提案的主要主题是：1)DNA聚合酶增量异四聚体及其亚组分的表征，以及为了了解哺乳动物复制复合体的结构而鉴定新的辅助蛋白，2)PolDelta与增殖细胞核抗原之间相互作用的表征，以及3)控制细胞DNA复制的调控系统与PolDelta之间的分子联系的研究。这些研究建立在我们实验室以前和现在的工作基础上，在此期间，我们开发了工具和必要的专业知识。我们将使用重组的polDelta异四聚体作为我们研究的平台，其中包括我们发现的两个新的亚基。我们将研究亚基的分子组织，以及复合体与增殖细胞核抗原的物理和功能相互作用。将确定polDelta的各个亚基对增殖细胞核抗原结合的贡献。将进一步提纯一种高分子形式的POL三角洲，以鉴定与POL三角洲异四聚体相关的蛋白质(S)。与p50亚基相互作用的两种新蛋白p38和p46将被研究为polDelta的可能辅助蛋白/亚基。将通过确定p38和p46是否可以重组为polDelta复合体来分析p38和p46与polDelta的相互作用。它们的潜在功能将通过生物化学、分子生物学和细胞生物学相结合的方法进行研究。我们将研究活体中polDelta酶亚基的磷酸化对细胞周期的依赖性，并将确定细胞周期蛋白/CDKs作为中介激酶的作用。标记亚基的胰酶多肽图谱将被用来识别其磷酸化水平a)依赖于细胞周期和b)被Cyclin/cdk抑制剂抑制的位点。我们将研究参与细胞周期检查点信号转导的蛋白激酶对polDelta的磷酸化作用。我们将研究POL三角洲磷酸化的功能后果。这些研究旨在确定p125是否是这些激酶的细胞靶点，研究DNA损伤过程中p125在体内的磷酸化，以及检测polDelta的磷酸化的功能后果。将研究p68作为靶向蛋白在将蛋白磷酸酶-1募集到polDelta复合体中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proposed studies represent a continuation of our long term goals of understanding the enzymology and functions of human DNA polymerase delta, a central enzyme in mammalian chromosomal DNA replication. The broad themes of the proposal are 1) the characterization of the DNA polymerase delta heterotetramer and its subassemblies, and the identification of novel accessory proteins with the goal of understanding the architecture of the mammalian replication complex, 2) the characterization of the interactions between pol delta and PCNA, and 3) the investigation of the molecular linkages between regulatory systems that control cellular DNA replication and pol delta. These studies build on previous and current work in our laboratory during which time we have developed the tools and necessary expertise. We will use as a platform for our studies the reconstituted pol delta heterotetramer which includes the two new subunits that we have discovered. We will investigate the molecular organization of the subunits, and the physical and functional interactions of the complex with PCNA. The contribution of individual subunits of pol delta to the binding of PCNA will be determined. A high molecular weight form of pol delta will be further purified to identify the protein(s) that associate with the pol delta heterotetramer. Two novel proteins, p38 and p46, that interact with the p50 subunit, will be studied as putative accessory proteins/subunits of pol delta. The interaction of p38 and p46 with pol delta will be analyzed by determining if they can be reconstituted into pol delta complexes. Their potential functions will be investigated by a combination of biochemical, molecular biological and cell biological approaches. The cell cycle dependence of the phosphorylation of the subunits of the pol delta enzyme in vivo will be investigated, and the role of the cyclin/cdks as the mediator kinases will be established. Tryptic peptide mapping of labeled subunits will be used to identify those sites whose phosphorylation levels are a) cell cycle dependent and b) inhibited by cyclin/cdk inhibitors. We will investigate phosphorylation of pol delta by the protein kinases involved in cell cycle checkpoint signaling. Functional consequences of phosphorylation of pol delta will be investigated. These studies are directed to establishing if p125 is a cellular target of these kinases, investigation of the in vivo phosphorylation of p125 during DNA damage, and examination the functional consequences of phosphorylation of pol delta. The role of p68 as a targeting protein for the recruitment of protein phosphatase-1 to the pol delta complex will be investigated.