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

基本信息

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
项目类别：
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

项目摘要

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 Pol α-primase 合成 7-10 个核苷酸的 RNA 引物，然后 Pol α-primase 延伸该引物在解离前用额外的 10-20 dNMP 进行引物 Pol α-引物酶由四个亚基组成，包括 Pri1 和 Pri2 引物酶亚基、催化 Pol1 亚基和必需的 Pol12 调节亚基芽殖酵母复制解旋酶，由 Cdc45、Mcm2-7 和 GINS（CMG 解旋酶）组成，我们的初步数据证明了复制叉之间重要的、新颖的相互作用。 Pol α-引物酶的 Pol12 亚基与 Mcm2-7 或单链 DNA 之间还存在相互作用。 Pol12 和 Mcm2-7 通过 S 期细胞周期蛋白依赖性激酶 (S-CDK) 功能得到增强。作为肿瘤标志物，Pol α-引物酶会在基因组中产生 DNA 突变，而 S-CDK 目前是因此，我们可以从机制上了解 Pol12 亚基如何治疗癌症。 Pol α-引物酶与解旋酶、细胞周期激酶和 DNA 协调发挥作用，可能导致改善癌症的诊断或预防方式。我们将首先确定在复制启动过程中 Pol α-引物酶如何被招募到 CMG 解旋酶中。我在实验室中使用纯化的芽殖酵母蛋白重建了 DNA 复制起始测定，使用我发现Pol12与Mcm2-7结合，并且我已经鉴定出点突变。 Pol12 特异性破坏与 Mcm2-7 的相互作用，该突变体的表达对酵母细胞是致命的，并且，我们将测试以下假设：Pol12-Mcm2-7 相互作用是 Pol α-引物酶招募所需的使用体外和体内方法在复制起始过程中使用 CMG 解旋酶。我们还将确定 Pol α-引物酶在复制起始过程中如何被激活以合成 RNA。发现 Pol12 在体外与 ssDNA 结合，并且我还确定 Pol12-ssDNA 相互作用是我们将测试 Pol12 与 ssDNA 相互作用是 Pol α-primase 所必需的假设。使用复制起始系统进行体外 RNA 合成活性，使用条件降解决定子进行体内 RNA 合成活性拉紧。确定 Pol α-引物酶如何被招募至 CMG，Pol α-引物酶 RNA 合成如何激活，Pol12 亚基的基本功能将揭示对如何 CMG 解旋酶与 Pol α 聚合酶偶联以启动真核生物中的 DNA 合成。我们对真核细胞复制如何提前启动的理解，并可能最终导致进一步的研究该提案还将提供出色的培训。研究生和本科生的机会。！