The Initiation of DNA Replication in Eukaryotes

真核生物中 DNA 复制的起始

• 批准号: 9381198
• 负责人: Yanchang Wang
• 金额: $ 29.67万
• 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: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9381198
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Binding; Binding Proteins; Biochemical; Biological Assay; Cell Cycle; DNA; DNA Binding; DNA biosynthesis; DNA replication fork; Data; Defect; Development; Diagnosis; Dissociation; Eukaryota; Event; Exclusion; Future; G1 Phase; Genomic DNA; Hand; In Vitro; Malignant Neoplasms; Mediating; Mitosis; Motor; Peptide Initiation Factors; Phosphorylation; Phosphorylation Site; Phosphotransferases; Polymerase; Process; Proteins; Replication Initiation; Replication Origin; S Phase; Saccharomycetales; Single-Stranded DNA; Site-Directed Mutagenesis; Testing; Tumor Markers; cancer biomarkers; cancer diagnosis; cancer therapy; cell growth; chemotherapeutic agent; chemotherapy; ds-DNA; experimental study; helicase; in vivo; melting; mutant; outcome forecast; prevent; reconstitution; yeast protein

Abstract The replication fork helicase unwinds genomic DNA at a replication fork. The assembly and activation of the eukaryotic replication fork helicase is highly regulated. Cdc45, Mcm2-7, and GINS (CMG) form a large assembly that is the active helicase, and the Mcm2-7 is the heterohexameric ATPase that forms the motor of the CMG. The assembly and activation of the CMG is governed by two essential S-phase kinases (S-CDK and DDK), and four essential initiation factors (Sld2, Sld3, Dpb11, and Mcm10) in budding yeast. S-CDK and DDK are currently investigated as targets for the development of cancer chemotherapeutic agents, and Mcm2-7 proteins serve as tumor markers. The Mcm2-7 loads as a double hexamer in late M and G1 phases, and in S phase the Mcm2-7 rings dissociates to single hexamers (Figure 1). Critical unanswered question in the initiation of DNA replication are: (1) How is the Mcm2-7 ring opened during S phase to allow for the extrusion of ssDNA (i.e. origin melting)? (2) How is origin DNA melted? and (3) How is melted origin DNA transferred to RPA, the eukaryotic single-stranded binding protein? Our central hypotheses are that DDK and S-CDK activity function with the essential initiation factors, Sld2, Sld3, Dpb11 and Mcm10, to open the Mcm2-7 ring, melt origin DNA, stabilize melted origin single-stranded DNA, and transfer melted origin DNA to RPA. We have also reconstituted a DNA replication initiation assay using purified budding yeast proteins, and we have generated or acquired conditional degron strains for each of the replication proteins. Thus, we will use a combination of in vitro reconstitution assays and in vivo experiments to test our hypotheses. We will first determine the Mcm2-7 subunit interface required for origin melting during S phase. We will also determine whether Mcm2-7 ring opening is required for subsequent CMG assembly or Mcm2-7 double-hexamer dissociation, or whether CMG assembly and double-hexamer dissociation occur prior to Mcm2-7 ring opening. Thus, we will establish the sequence of key events required for DNA replication initiation. We will also test the hypothesis that S-CDK and DDK phosphorylate Mcm2-7 proteins to promote origin melting during S phase. Sld2, Sld3, Dpb11, or Mcm10 each has biochemical activity for binding origin ssDNA. We will determine how Sld2, Sld3, Dpb11, and Mcm10 function with one another to stabilize single-stranded DNA as it is produced during the process of origin melting. Our hypothesis is that Mcm10 or Sld2-Sld3-Dpb11 function in a S-CDK-dependent manner coordination to stabilize melted origin DNA, preventing reannealing to double-stranded DNA. Finally, we will determine how the melted origin DNA is ultimately transferred to RPA. We have preliminary data suggesting that Dpb11 interaction with RPA is required for DNA replication, and we propose that Dpb11 hands-off melted single-stranded DNA to RPA at a replication origin. Taken together, these three aims will provide a comprehensive view of how cell cycle kinases function with replication initiator proteins to mediate replication fork helicase activation and DNA replication initiation in eukaryotes.

抽象的 复制叉解旋酶在复制叉处解开基因组 DNA。组装和激活 真核复制叉解旋酶的功能受到高度调控。 Cdc45、Mcm2-7 和 GINS (CMG) 组成了一个大的 组装体是活性解旋酶，Mcm2-7 是异六聚体 ATP 酶，形成解旋酶的马达 CMG。 CMG 的组装和激活由两种重要的 S 期激酶（S-CDK 和 DDK），以及芽殖酵母中的四种必需起始因子（Sld2、Sld3、Dpb11 和 Mcm10）。 S-CDK和DDK 目前正在作为癌症化疗药物开发的靶点进行研究，Mcm2-7 蛋白质充当肿瘤标志物。 Mcm2-7 在 M 后期和 G1 阶段以及 S 阶段作为双六聚体加载 Mcm2-7 环解离为单个六聚体（图 1）。关键的未回答问题 DNA复制的起始点是：（1）Mcm2-7环如何在S期打开以允许挤出 ssDNA（即起源熔化）？ (2) 原始DNA是如何融化的？ (3) 熔化的起始 DNA 如何转移至 RPA，真核单链结合蛋白？我们的中心假设是 DDK 和 S-CDK 活性 与必需的引发因子 Sld2、Sld3、Dpb11 和 Mcm10 一起作用，打开 Mcm2-7 环，熔化 起始DNA，稳定熔化的起始单链DNA，并将熔化的起始DNA转移至RPA。 我们还使用纯化的芽殖酵母蛋白重建了 DNA 复制起始测定，并且 我们已经为每个复制蛋白生成或获得了条件降解决定子菌株。这样，我们将 结合体外重构测定和体内实验来检验我们的假设。我们首先会 确定 S 相期间原点熔化所需的 Mcm2-7 亚基界面。我们还将确定 后续CMG组装或Mcm2-7双六聚体是否需要Mcm2-7开环 解离，或者 CMG 组装和双六聚体解离是否发生在 Mcm2-7 环打开之前。 因此，我们将建立 DNA 复制起始所需的关键事件序列。 我们还将检验 S-CDK 和 DDK 磷酸化 Mcm2-7 蛋白以促进起源的假设 S相熔化。 Sld2、Sld3、Dpb11 或 Mcm10 均具有结合原始 ssDNA 的生化活性。 我们将确定 Sld2、Sld3、Dpb11 和 Mcm10 如何相互发挥作用以稳定单链 DNA 在起源熔化过程中产生。我们的假设是 Mcm10 或 Sld2-Sld3-Dpb11 以 S-CDK 依赖性方式协调发挥作用，以稳定熔化的起始 DNA，防止重新退火 双链DNA。最后，我们将确定如何将熔化的原始 DNA 最终转移至 RPA。 我们有初步数据表明 Dpb11 与 RPA 的相互作用是 DNA 复制所必需的，并且我们 提出 Dpb11 在复制起点处将熔化的单链 DNA 交给 RPA。综合起来， 这三个目标将提供细胞周期激酶如何与复制启动子一起发挥作用的全面视图 介导真核生物复制叉解旋酶激活和 DNA 复制起始的蛋白质。