Mechanism of DNA replication initiation in Saccharomyces cerevisiae

描述（由申请人提供）： 摘要 准确的 DNA 复制对于所有生物体细胞增殖过程中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 DNA 复制的复杂性曾困扰着直接解决体外这一过程涉及的生化机制的努力。我们开发了功能性体外测定，使我们能够克服这一限制。我们之前已经证明，预复制复合物（pre-RCs）允许G1期的起源，随后在S期激活，可以用从芽殖酵母、酿酒酵母中纯化的蛋白质来重建。重要的是，我们最近发现重建的前 RC 也支持体外受调节的 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处理的真核 DNA 复制起始中的关键问题，形成此处提出的实验的基础：目标 1) Mcm2-7 由 G1 期的前 RC 以非活性形式加载到起点上。 Mcm2-7 解旋酶的激活以及伴随的 S 期复制体组装需要在 pre-RC 周围形成预起始复合物 (pre-IC)。目的是利用纯化的芽殖酵母蛋白进行重构研究，描绘预 IC 的起源激活机制。我们将描述预 IC 组装的顺序，确定预 IC 子组件的结构，定义诱导局部原点解旋的步骤，并将生化功能分配给预 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。目标 2) 在解旋酶激活之前，Mcm2-7 复合物可以被动地沿着 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，这对真核起源规范机制具有重要意义。激活后，Mcm2-7 复合物以定向方式主动沿 DNA 易位，在分叉处展开 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 Mcm2-7 解旋酶复合物的结构构型，并确定相反方向的 Mcm2-7 六聚体在延伸过程中是否物理分离或保持结合。这些研究将深入了解 Mcm2-7 DNA 解旋机制，该机制是真核复制体组织的基础。目标 3) 我们之前发现两个 Mcm2-7 六聚体通过 pre-RC 以相反的方向协同加载在双链 DNA 周围。为了阐明 Mcm2-7 加载的机制，我们将在体外和体内使用位点特异性蛋白质-蛋白质交联来定义预 RC 组装过程中 Cdc6 的相互作用网络。这种方法将 两者都确定了 pre-RC 中 Cdc6 的功能相互作用伙伴，并为 pre-RC 的结构模型提供了信息。

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.