Function and mechanism of the essential, Pol12 subunit of the eukaryotic Pol alpha-primase

真核生物 Pol α-引物酶必需的 Pol12 亚基的功能和机制

• 批准号: 9304633
• 负责人: Irina Bruck
• 金额: $ 8.17万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2017-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9304633
• 关键词: Binding; Biological Assay; Cell Cycle; Cell Survival; Cell division; Cells; Couples; Cyclin-Dependent Kinases; DNA; DNA Primase; DNA Sequence; DNA Sequence Alteration; DNA Structure; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Dependence; Development; Diagnosis; Disease; Dissociation; Eukaryota; Eukaryotic Cell; Generations; Genome; Human; Hybrids; In Vitro; Laboratories; Lead; Length; MCM Protein; Malignant Neoplasms; Methodology; N-terminal; Nucleic Acids; Nucleotides; Organism; Phosphotransferases; Point Mutation; Polymerase; Process; Proteins; Protocols documentation; RNA; RNA chemical synthesis; RNA primers; Reagent; Recruitment Activity; Replication Initiation; Role; S Phase; Saccharomycetales; Single-Stranded DNA; System; Testing; Tumor Markers; Yeasts; base; cancer diagnosis; cancer therapy; chemotherapy; graduate student; helicase; in vitro activity; in vivo; mutant; novel; outcome forecast; prevent; protein function; protein protein interaction; reconstitution; small molecule inhibitor; training opportunity; undergraduate student; yeast protein

Project Abstract The Pol α-primase synthesizes a 7-10 nucleotide RNA primer, and Pol α-primase then extends this primer by an additional 10–20 dNMP before dissociating. Pol α-primase is comprised of four subunits, including the Pri1 and Pri2 primase subunits, the catalytic Pol1 subunit, and the essential Pol12 regulatory subunit. The budding yeast replicative helicase, comprised of Cdc45, Mcm2-7, and GINS (CMG helicase), unwinds DNA at a replication fork. Our preliminary data demonstrate essential, novel interactions between the Pol12 subunit of Pol α-primase and Mcm2-7 or single-stranded DNA. Furthermore, the interaction between Pol12 and Mcm2-7 is strengthened by the S phase cyclin-dependent kinase (S-CDK). Mcm proteins function as tumor markers, Pol α-primase generates DNA mutations in the genome, and S-CDK is currently a target for small molecule inhibitors to treat cancer. Thus, a mechanistic understanding of how the Pol12 subunit of the Pol α-primase functions in coordination with the helicase, cell-cycle kinase, and DNA may lead to improvements in how cancer is diagnosed or prevented. We will first determine how Pol α-primase is recruited to the CMG helicase during replication initiation. I reconstituted the DNA replication initiation assay in my laboratory using purified budding yeast proteins, using reagents produced in my laboratory. I found that Pol12 binds to Mcm2-7, and I have identified point mutations of Pol12 that specifically disrupt the interaction with Mcm2-7. Expression of this mutant is lethal to yeast cells, and, we will test the hypothesis that Pol12-Mcm2-7 interaction is required for Pol α-primase recruitment to CMG helicase during replication initiation using the in vitro and in vivo methodologies. We will also determine how Pol α-primase is activated to synthesize RNA during replication initiation. I found that Pol12 binds to ssDNA in vitro, and I also determined that Pol12-ssDNA interaction is required for yeast cell viability. We will test the hypothesis that Pol12 interaction with ssDNA is required for Pol α-primase RNA synthesis activity in vitro, using the replication initiation system, and in vivo, using a conditional degron strain. Determining how Pol α-primase is recruited to the CMG, how Pol α-primase RNA synthesis is activated, and the essential function for the Pol12 subunit will reveal a mechanistic understanding of how the CMG helicase couples to the Pol α polymerase to initiate DNA synthesis in eukaryotes. This project will advance our understanding of how replication is initiated in eukaryotic cells, and may ultimately lead to further advances in the prognosis and treatment of cancer. This proposal will also provide excellent training opportunities for graduate and undergraduate students. !

项目摘要 polα-蛋白酶合成了7-10个核苷酸RNA底漆，然后Polα-蛋白酶扩展了这一点 解离之前，底漆再增加10–20 DNMP。 polα-蛋白酶完成了四个亚基， 包括PRI1和PRI2原始酶亚基，催化POL1亚基以及必需的POL12调节 亚基。发芽的酵母复制型解旋酶，包括CDC45，MCM2-7和杜松子（CMG解旋酶）， 在复制叉上放开DNA。我们的初步数据表明 POL1-POLα-原酶和MCM2-7或单链DNA的POL12亚基。此外， POL12和MCM2-7通过S相依赖性激酶（S-CDK）增强。 MCM蛋白质功能 作为肿瘤标记，polα-蛋白酶在基因组中产生DNA突变，而S-CDK当前是一个目标 小分子抑制剂治疗癌症。这是对如何使用Pol12亚基的机械理解 polα-原则酶在与解旋酶，细胞周期激酶和DNA协调中的功能可能导致 改善癌症的诊断或预防方式。 我们将首先确定如何在复制计划期间将polα-蛋白酶募集到CMG解旋酶。 我使用纯化的芽 我的实验室生产的试剂。我发现POL12与MCM2-7结合，并且已经确定了点突变 POL12的特异性破坏了与MCM2-7的相互作用。该突变体的表达对酵母细胞是致命的， 而且，我们将检验以下假设：POL12-MCM2-7相互作用需要POLα-原酶募集需要 使用体外和体内方法复制启动过程中的CMG解旋酶。 我们还将确定如何激活polα-蛋白酶在复制启动过程中合成RNA。我 发现POL12在体外与ssDNA结合，我还确定POL12-SSDNA相互作用是需要的 酵母细胞活力。我们将检验以下假设：POL1-POLα-蛋白酶需要POL12与ssDNA相互作用 使用有条件的DEGRON，使用复制启动系统和体内的RNA合成活性，并在体内 拉紧。 确定如何募集polα-蛋白酶为CMG，Polα-原则RNA合成是如何募集的 被激活，POL12亚基的基本功能将揭示对如何如何 CMG解旋酶伴侣与polα聚合酶一起启动真核生物中的DNA合成。这个项目将 促进我们对真核细胞中如何启动复制的理解，并最终可能导致进一步 癌症预后和治疗的进步。该建议还将提供出色的培训 研究生和本科生的机会。 呢