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.

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 真核复制叉解旋酶受高度调节。 CDC45，MCM2-7和杜松子酒（CMG）形成大型 组装是活性解旋酶，MCM2-7是构成电动机的杂项ATPase CMG。 CMG的组装和激活由两个必需的S期激酶（S-CDK和 DDK）和四个基本的起始因子（SLD2，SLD3，DPB11和MCM11）在发芽的酵母中。 S-CDK和DDK 目前被研究为开发癌症化学治疗剂的靶标，MCM2-7 蛋白质用作肿瘤标记。 MCM2-7在晚期和G1阶段的双重六聚体中加载，在S 阶段MCM2-7环分离为单个六聚体（图1）。关键的未解决问题 DNA复制的启动是：（1）如何在S相期间打开MCM2-7环以进行挤压 ssDNA（即起源熔化）？ （2）原点DNA如何融化？ （3）如何转移融化的原点DNA到 RPA，真核单链结合蛋白？我们的中心假设是DDK和S-CDK活动 与基本启动因子（SLD2，SLD3，DPB11和MCM11）一起运行MCM2-7环，熔体的功能 原点DNA，稳定融化的单链DNA，并将融化的原点DNA转移到RPA。 我们还使用纯化的芽酵母蛋白重构了DNA复制启动测定法，并且 我们已经为每种复制蛋白生成或获得有条件的DEGRON菌株。因此，我们会的 使用体外重构测定和体内实验的组合来检验我们的假设。我们将首先 确定在S相期间原点熔化所需的MCM2-7亚基界面。我们还将确定 随后的CMG组件还是MCM2-7 Double-Hexamer，MCM2-7是否需要打开戒指 解离，还是CMG组装和双己糖离解之前发生在MCM2-7环开口之前。 因此，我们将建立DNA复制启动所需的关键事件的顺序。 我们还将检验以下假设：S-CDK和DDK磷酸化MCM2-7蛋白以促进起源 在s阶段熔化。 SLD2，SLD3，DPB11或MCM10每个具有结合起源ssDNA的生化活性。 我们将确定SLD2，SLD3，DPB11和MCM10如何互相稳定单链 在原点熔化过程中产生的DNA。我们的假设是MCM10或SLD2-SLD3-DPB11 以S-CDK依赖性方式协调功能，以稳定融化的原点DNA，以防止重新进行 双链DNA。最后，我们将确定如何最终将融化的原点DNA转移到RPA。 我们有初步数据，表明DPB11与RPA相互作用是DNA复制所需的，而我们 提出DPB11的解雇将单链DNA融化为复制起源的RPA。在一起， 这三个目标将为细胞周期激酶如何通过复制引发剂发挥作用提供全面的看法 蛋白质以介导真核生物中的复制叉解旋酶激活和DNA复制起始。