The Initiation of DNA Replication in Eukaryotes

真核生物中 DNA 复制的起始

• 批准号: 9749991
• 负责人: Yanchang Wang
• 金额: $ 29.83万
• 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/9749991
• 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.

摘要 复制叉解旋酶在复制叉处解旋基因组DNA。组装和激活 真核生物复制叉解旋酶的活性受到高度调节。Cdc 45、Mcm 2 -7和GINS（CMG）形成了一个大的 Mcm 2 -7是异源六聚体ATP酶，形成了 CMG。CMG的组装和激活由两种必需的S期激酶（S-CDK和 DDK）和四个必需的起始因子（Sld 2、Sld 3、Dpb 11和Mcm 10）。S-CDK和DDK 目前被研究作为癌症化疗剂开发的靶点，Mcm 2 -7 蛋白质作为肿瘤标志物。Mcm 2 -7在M期和G1期晚期作为双六聚体加载，在S期作为双六聚体加载。 在第一阶段，Mcm 2 -7环解离成单个六聚体（图1）。关键的未回答的问题， DNA复制的起始是：（1）Mcm 2 -7环在S期是如何打开以允许挤出的 ssDNA（即起源熔化）？(2)起源DNA是如何融化的？以及（3）熔化的起源DNA是如何转移到 RPA，真核单链结合蛋白？我们的中心假设是DDK和S-CDK活性 与必需的起始因子Sld 2、Sld 3、Dpb 11和Mcm 10一起起作用，打开Mcm 2 -7环，解链 起始DNA，稳定解链的起始单链DNA，并将解链的起始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， 我们已经产生或获得了每种复制蛋白的条件降解决定子菌株。因此，我们将 使用体外重建测定和体内实验的组合来测试我们的假设。我们将首先 确定S期起始熔解所需的Mcm 2 -7亚基界面。我们还将确定 后续CMG组装需要Mcm 2 -7开环还是Mcm 2 -7双六聚体 在Mcm 2 -7开环之前是否发生CMG组装和双六聚体解离。 因此，我们将建立DNA复制启动所需的关键事件序列。 我们还将检验S-CDK和DDK磷酸化Mcm 2 -7蛋白以促进起源的假设。 在S阶段熔化。Sld 2、Sld 3、Dpb 11或Mcm 10各自具有结合起始ssDNA的生物化学活性。 我们将确定Sld 2，Sld 3，Dpb 11和Mcm 10如何相互作用以稳定单链 DNA是在起源熔化过程中产生的。我们的假设是Mcm 10或Sld 2-Sld 3-Dpb 11 以S-CDK依赖性方式协调稳定解链的起始DNA，防止再退火， 双链DNA最后，我们将确定熔化的原始DNA最终如何转移到RPA。 我们有初步的数据表明，Dpb 11与RPA的相互作用是DNA复制所必需的，我们 提出Dpb 11在复制起点处将单链DNA不经手地熔化到RPA。综合起来看， 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观点。 真核生物中介导复制叉解旋酶激活和DNA复制起始的蛋白质。