Mechanism of DNA replication initiation in Saccharomyces cerevisiae

酿酒酵母 DNA 复制起始机制

• 批准号: 9120378
• 负责人: Dirk Remus
• 金额: $ 42.22万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9120378
• 关键词: Address; Amino Acids; Architecture; Biochemical; Biochemistry; Biological Assay; Cell Cycle; Cell Proliferation; Cells; Chromosomal Rearrangement; Chromosomes; Competence; Complex; DNA; DNA Replication Factor; DNA biosynthesis; DNA replication fork; Defect; Development; Electron Microscopy; Ensure; Eukaryota; Exhibits; Fluorescence Microscopy; G1 Phase; Genome; Genome Stability; Goals; Head; Health; In Vitro; Lead; Length; Licensing; Maintenance; Malignant Neoplasms; Maps; Molecular; Mutation; Organism; Peptide Initiation Factors; Positioning Attribute; Pre-Replication Complex; Process; Proteins; Reaction; Recombinants; Regulation; Replication Initiation; Replication Origin; Role; S Phase; Saccharomyces cerevisiae; Saccharomycetales; Site; Slide; Specificity; Staging; Structural Models; Structure; Surface; System; Testing; Time; Yeasts; base; crosslink; ds-DNA; genetic information; helicase; human disease; in vitro Assay; in vivo; insight; interdisciplinary approach; interest; novel; prevent; protein complex; protein purification; protein reconstitution; reconstitution; research study; single molecule; tool; yeast protein

DESCRIPTION (provided by applicant): Summary Accurate DNA replication is essential for the maintenance of the genetic information during cell proliferation in all organisms. Defects in DNA replication factors are associated with human diseases including cancer. The complexity of eukaryotic DNA replication has previously confounded efforts to directly address biochemical mechanisms involved in this process in vitro. We have developed functional in vitro assays that allow us to overcome this limitation. We have shown previously that pre-replicative complexes (pre-RCs), which license origins in G1 phase for subsequent activation in S phase, can be reconstituted with proteins purified from budding yeast, Saccharomyces cerevisiae. Importantly, we recently found that reconstituted pre-RCs also support regulated DNA replication in vitro, exhibiting the fundamental hallmarks of cellular DNA replication. These functional assays enable us to use interdisciplinary approaches to address key problems in eukaryotic DNA replication initiation that were previously intractable, forming the basis for experiments proposed here: Aim 1) Mcm2-7 is loaded onto origins in inactive form by the pre-RC in G1 phase. Activation of the Mcm2-7 helicase, and concomitant replisome assembly in S phase, requires the formation of a pre-initiation complex (pre-IC) around the pre-RC. The goal is to delineate the mechanism of origin activation by the pre-IC using reconstitution studies with purified budding yeast proteins. We will characterize the order of pre-IC assembly, determine the structure of pre-IC subassemblies, define the steps that induce local origin unwinding, and assign biochemical functions to pre-IC components. These studies form the basis for our long-term goal to reconstitute the entire eukaryotic DNA replication reaction in vitro. Aim 2) Mcm2-7 complexes can passively slide along DNA prior to activation of the helicase. We will characterize this sliding ability and test its role in initiatin site determination, which has important implications for the eukaryotic origin specification mechanism. Upon activation, Mcm2-7 complexes actively translocate along DNA in a directed manner to unwind DNA at the fork. We will use biochemistry, electron microscopy, and advanced single molecule fluorescence microscopy to analyze the structural configuration of active Mcm2-7 helicase complexes and determine if Mcm2-7 hexamers of opposite orientation physically separate or remain associated during elongation. These studies will provide insight into the Mcm2-7 DNA unwinding mechanism, which underlies eukaryotic replisome organization. Aim 3) We have found previously that two Mcm2-7 hexamers are cooperatively loaded in opposite orientation around double stranded DNA by the pre-RC. To elucidate the mechanism for Mcm2-7 loading we will define the interaction network of Cdc6 during pre-RC assembly using site-specific protein-protein cross-linking in vitro and in vivo. This approach will both identify the functional interaction partners for Cdc6 in the pre-RC and inform structural models for the pre-RC.

描述(由申请人提供)： 摘要在所有生物体的细胞增殖过程中，准确的DNA复制对于维持遗传信息是必不可少的。DNA复制因子的缺陷与包括癌症在内的人类疾病有关。真核DNA复制的复杂性之前已经扰乱了在体外直接解决这一过程所涉及的生化机制的努力。我们已经开发出功能性体外分析方法，使我们能够克服这一限制。我们以前已经证明，复制前复合体(Pre-RCS)可以与从萌芽酵母中纯化的蛋白质重组，该复合体允许起始于G1期，随后在S期激活。重要的是，我们最近发现，重组的Pre-RCs也支持体外受调控的DNA复制，表现出细胞DNA复制的基本特征。这些功能分析使我们能够使用跨学科的方法来解决真核DNA复制启动中的关键问题，这些问题以前是难以解决的，为这里提出的实验奠定了基础：目的1)Mcm2-7在G1期由前RC以非活性的形式加载到起点上。McM2-7解旋酶的激活，以及伴随的S阶段的复制体组装，需要在前RC周围形成一个预启动复合体(PreIC)。目的是通过对纯化的发芽酵母蛋白的重组研究来描述前IC的来源激活机制。我们将确定Pre-IC组装的顺序，确定Pre-IC子组件的结构，定义诱导局部原点解开的步骤，并为Pre-IC组件分配生化功能。这些研究为我们在体外重建整个真核DNA复制反应的长期目标奠定了基础。目的2)McM2-7复合体可以在解旋酶激活之前被动地沿DNA滑动。我们将表征这种滑动能力，并测试其在起始位点确定中的作用，这对真核细胞起源指定机制具有重要意义。激活后，Mcm2-7复合体以定向的方式主动沿DNA移位，在分叉处解开DNA。我们将使用生化、电子显微镜和先进的单分子荧光显微镜来分析活性McM2-7解旋酶复合体的结构构型，并确定在伸长过程中相反取向的McM2-7六聚体是物理上分离的还是保持联系的。这些研究将提供对Mcm2-7 DNA解离机制的洞察，该机制是真核复制体组织的基础。目的3)我们先前已经发现，两个McM2-7六聚体被前RC以相反的方向协同负载在双链DNA周围。为了阐明Mcm2-7的负载机制，我们将在体内外利用特定部位的蛋白质-蛋白质交联来定义前RC组装过程中CDC6的相互作用网络。这一方法将 两者都确定了Pre-RC中CDC6的功能交互伙伴，并为Pre-RC提供了结构模型。