Mechanism of DNA replication initiation in Saccharomyces cerevisiae

酿酒酵母 DNA 复制起始机制

• 批准号: 9332388
• 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/9332388
• 关键词: Address; Alpha Cell; 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; In Vitro; Lead; Length; Licensing; Maintenance; Malignant Neoplasms; Maps; Molecular; Multiprotein Complexes; 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; Structural Models; Structure; Surface; System; Testing; Time; Yeasts; crosslink; ds-DNA; experimental study; genetic information; helicase; human disease; in vitro Assay; in vivo; insight; interdisciplinary approach; interest; novel; prevent; protein purification; protein reconstitution; public health relevance; reconstitution; 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）： 在所有生物体中，准确的DNA复制对于细胞增殖期间遗传信息的维持至关重要。DNA复制因子的缺陷与包括癌症在内的人类疾病有关。真核生物DNA复制的复杂性以前混淆了直接解决在体外这一过程中涉及的生化机制的努力。我们已经开发了功能性体外试验，使我们能够克服这一限制。我们以前已经表明，前复制复合物（前RC），许可证的起源在G1期为随后的激活在S期，可以重建与蛋白质纯化的芽殖酵母，酿酒酵母。重要的是，我们最近发现，重组前RC也支持体外调控的DNA复制，表现出细胞DNA复制的基本特征。这些功能测定使我们能够使用跨学科的方法来解决以前难以解决的真核DNA复制起始中的关键问题，形成了本文提出的实验的基础：目的1）Mcm 2 -7在G1期通过pre-RC以非活性形式加载到起点上。Mcm 2 -7解旋酶的激活以及伴随的S期复制体组装需要在前RC周围形成前起始复合物（前IC）。我们的目标是描绘的起源激活的机制，前IC使用纯化的芽殖酵母蛋白质的重建研究。我们将描述前IC组装的顺序，确定前IC的结构，定义诱导局部起源解旋的步骤，并将生化功能分配给前IC组件。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）Mcm 2 -7复合物能在解旋酶激活前沿着DNA被动滑动。我们将描述这种滑动能力，并测试其在起始位点确定中的作用，这对真核生物的起源特化机制具有重要意义。在激活时，Mcm 2 -7复合物以定向方式沿着沿着DNA主动易位，以在分叉处解开DNA。我们将使用生物化学，电子显微镜和先进的单分子荧光显微镜来分析活性Mcm 2 -7解旋酶复合物的结构构型，并确定Mcm 2 -7相反方向的六聚体在延伸过程中是否物理分离或保持关联。这些研究将提供深入了解Mcm 2 -7 DNA解旋机制，这是真核生物复制体组织的基础。目的3）我们以前已经发现两个Mcm 2 -7六聚体通过pre-RC以相反的方向协同装载在双链DNA周围。为了阐明Mcm 2 -7加载的机制，我们将在体外和体内使用位点特异性蛋白质-蛋白质交联来定义Cdc 6在预RC组装期间的相互作用网络。这种方法将 两者都确定了前RC中Cdc 6的功能相互作用伙伴，并为前RC提供了结构模型。