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.

摘要 复制叉解旋酶在复制叉处解开基因组DNA。组装和激活 真核生物的复制分叉解旋酶是高度调控的。Cdc45、McM2-7和GINS(CMG)形成了一个大的 组装是活性解旋酶，而McM2-7是形成马达的异六聚体ATPase。 CMG。CMG的组装和激活受两个必需的S相激酶(S-CDK和 DDK)和四种基本启动因子(SLD2、SLD3、Dpb11和Mcm10)在芽殖酵母中表达。S--CDK与DDK 目前被研究为开发癌症化疗药物的靶点，以及Mcm2-7 蛋白质充当肿瘤标记物。McM2-7在M期和G1期晚期以双六角体形式存在，在S 相：Mcm2-7环解离为单六聚体(图1)。中的关键未解答问题 DNA复制的启动是：(1)在S阶段，McM2-7环是如何打开的，以允许挤出 单链DNA(即起源融化)？(2)起源DNA如何融化？以及(3)融化的起源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 与基本的启动因子SLD2、SLD3、Dpb11和Mcm10一起作用，打开Mcm2-7环，融化 来源DNA，稳定融化的来源单链DNA，并将融化的来源DNA转移到RPA。 我们还用纯化的发芽酵母蛋白重建了DNA复制启动试验，并 我们已经为每个复制蛋白产生或获得了条件降解菌株。因此，我们将 使用体外重建试验和体内实验相结合的方法来检验我们的假设。我们将首先 确定S期原生融化所需的McM2-7亚基界面。我们还将确定 后续CMG装配或McM2-7双六角机是否需要打开Mcm2-7环 解离，或CMG组装和双六聚体解离是否发生在McM2-7开环之前。 因此，我们将建立启动DNA复制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期的熔融作用。SLD2、SLD3、Dpb11或Mcm10均具有结合起始单链DNA的生化活性。 我们将确定SLD2、SLD3、Dpb11和Mcm10如何相互作用以稳定单链 在原生物质融化过程中产生的DNA。我们的假设是Mcm10或SLd2-SLd3-Dpb11 功能以S-CDK依赖的方式协调稳定融化的起源dna，防止再退火到 双链DNA。最后，我们将确定融化的起源DNA最终是如何转移到RPA的。 我们有初步数据表明，DNA复制需要Dpb11与RPA的相互作用，我们 建议Dpb11在复制起始处将单链DNA熔化到RPA。加在一起， 这三个目标将提供细胞周期激酶如何在复制启动子中发挥作用的全面视图 真核生物中介导复制分叉解旋酶激活和dna复制启动的蛋白质。