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

项目摘要 Pol α-引物酶合成一个7-10个核苷酸的RNA引物，然后Pol α-引物酶将其延伸 在解离之前通过额外的10-20 dNMP对引物进行扩增。Pol α-引发酶由四个亚基组成， 包括Pri 1和Pri 2引发酶亚基、催化性Pol 1亚基和必需的Pol 12调节亚基。 亚单位芽殖酵母复制解旋酶由Cdc 45、Mcm 2 -7和GINS（CMG解旋酶）组成， 在复制叉处解开DNA我们的初步数据表明， Pol α-引发酶的Pol 12亚基和Mcm 2 -7或单链DNA。此外， Pol 12和Mcm 2 -7通过S期细胞周期蛋白依赖性激酶（S-CDK）而增强。MCM蛋白功能 作为肿瘤标志物，Pol α-引发酶在基因组中产生DNA突变，而S-CDK目前是 小分子抑制剂来治疗癌症。因此，对Pol 12亚基的机制的理解， Pol α-引发酶与解旋酶、细胞周期激酶和DNA协同作用可能导致 改善癌症的诊断或预防方法。 我们将首先确定在复制起始期间Pol α-引发酶如何被募集到CMG解旋酶。 我在我的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， 我实验室生产的试剂。我发现Pol 12与Mcm 2 -7结合， 的Pol 12，特异性地破坏与Mcm 2 -7的相互作用。这种突变体的表达对酵母细胞是致命的， 并且，我们将检验Pol 12-Mcm 2 -7相互作用是Pol α-引发酶募集所必需的假设， 使用体外和体内方法在复制起始期间的CMG解旋酶。 我们还将确定Pol α-引发酶在复制起始期间如何被激活以合成RNA。我 我发现Pol 12在体外与ssDNA结合，我还确定Pol 12-ssDNA相互作用是 酵母细胞活力。我们将检验Pol 12与ssDNA的相互作用是Pol α引发酶所必需的假设。 使用复制起始系统的体外RNA合成活性和使用条件降解决定子的体内RNA合成活性 株 确定Pol α-引发酶如何被募集到CMG，Pol α-引发酶RNA合成如何被激活。 激活，Pol 12亚基的基本功能将揭示一个机械的理解，如何激活， CMG解旋酶与Pol α聚合酶偶联以启动真核生物中的DNA合成。该项目将 推进我们对真核细胞中复制是如何启动的理解，并最终可能导致进一步的研究。 癌症的预后和治疗进展。这一建议还将提供出色的培训 研究生和本科生的机会。 !

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. !