Biochemistry of Eukaryotic Replication Fork and DNA Repair

项目摘要 DNA复制是由许多蛋白质执行的，这些蛋白质充当一个动态机器，称为复制体。这个 真核复制体的核心成分包括：1)一个11亚基的“CMG”解旋酶，它将 亲本DNA链，2)领先和滞后链DNA聚合酶(POL)，POL d和POL E， 分别为3)包裹DNA并将两个POL系在DNA上以获得高处理能力的增殖细胞核抗原滑动夹， 4)将增殖细胞核抗原装载到DNA上的RFC钳夹五聚体，以及制造杂交RNA的Pola-Primase- Pol启动DNA合成的DNA启动点。除了这些“核心”组件之外，还有 几种辅助蛋白包括RPA、Tof1、Mec1、Csm3、FACT、Mcm10、Ctf4、Ctf18-RFC。有一台主机 指在起始处围绕双链DNA组装两个CMG的蛋白质。CMG二聚体解开关闭的 未知反应中的双链和支架酶组装复制体。我们已经提纯了这些 酵母中的蛋白质(酿酒酵母；S.C.)系统。在这项建议中，我们会把研究范围扩展至 真核复制体的结构/功能。我们将使用生化和单分子方法来 确定增殖细胞核抗原是否如预期那样在滞后的链上积累，以及增殖细胞核抗原是否可以周期性地留在那里 在天真核小体错配修复和组装的前导链上。我们已经解决了无数 与我们的合作者李慧琳(密西西比州VanAndel研究所)合作，并有更多的结构正在进行中 并有计划。我们已经纯化了ATR DNA损伤检查点信号系统的几个因子 其中许多复制体蛋白是这一途径的靶标。我们计划进行生物化学研究，以澄清目标 以及它们对复制体的影响。我们将确定复制过程中核小体遗传的机制。 在后生动物中，核小体的表观遗传出了问题，可能导致癌症和其他疾病。在……里面 酵母，细胞研究表明，Mcm2组蛋白结合突变体可以防止表观遗传转移到滞后 缺乏Dpb3/4亚基的Pol e不会将表观遗传标记转移到领先的链上。我们 计划使用我们的单分子研究实时可视化复制过程中的核小体转移 新收购的Q Trap)与洛克菲勒大学的刘诗欣博士合作)。我们有各种各样的 核小体移动因子和具有不同荧光标记组蛋白的酵母核小体 学习。复制发生在核中心，拥有带有许多DNA复制叉子的“复制工厂”。我们的 最近的生化和结构研究确定了最基本的组成和原子结构 复制工厂的单位，二聚体复制体。我们将使用超分辨率显微镜来验证我们的 重组的工厂与内部的细胞是一样的。我们对起源的双链DNA是如何打开的有深入的了解 从我们最近的研究发现，环绕双链DNA的双链CMG解旋酶是 十年来，向内，与“向外”相反的方向思考。我们发现两个向内导向的CMG 可以将DNA切割成碎片。我们有计划进一步开展这方面的调查。

Project Summary DNA replication is performed by numerous proteins that act as a dynamic machine, termed a replisome. The core components of the eukaryotic replisome consist of 1) an 11 subunit “CMG” helicase that separates the parental DNA strands, 2) The leading and lagging strand DNA polymerases (Pol), Pol d and Pol e, respectively,3) The PCNA sliding clamp that encircles DNA and tethers both Pols to DNA for high processivity, 4) the RFC clamp loader pentamer that loads PCNA onto DNA and 5) Pol a-primase that makes a hybrid RNA- DNA primed site for the Pols to initiate DNA synthesis. In addition to these “core” components, there are several ancillary proteins including RPA, Tof1, Mec1, Csm3, FACT, Mcm10, Ctf4, Ctf18-RFC. There are a host of proteins that assemble two CMGs around duplex DNA at origins. The CMG dimer unwinds the closed duplex in an unknown reaction and scaffolds enzymes to assemble replisomes. We have purified these proteins in the yeast (Saccharomyces cerevisiae; S.c.) system. In this proposal, we will extend our studies on the structure/function of the eukaryotic replisome. We will use biochemical and single-molecule methods to determine if PCNA accumulates on the lagging strand as expected, and whether PCNA may periodically be left on the leading strand for mismatch repair and assembly of naïve nucleosomes. We have solved numerous structures with our collaborator, Huilin Li (VanAndel Institute, MI), and have many more structures in progress and planned. We have purified the several factors of the ATR DNA damage checkpoint signaling system of which many replisome proteins are targets of this pathway. We plan biochemical studies that will clarify targets and their effect on replisomes. We will determine the mechanism of nucleosome inheritance during replication. In metazoans, epigenetic inheritance of nucleosomes, gone awry, can lead to cancer and other diseases. In yeast, cell studies have shown that a Mcm2 histone binding mutant prevents epigenetic transfer to lagging strands, and Pol e lacking the Dpb3/4 subunits does not transfer epigenetic marks to the leading strand. We plan to visualize nucleosome transfer during replication in real time using single-molecule studies with our newly acquired Q trap) in collaboration with Dr. Shixin Liu (Rockefeller University)). We have various nucleosome mobility factors and yeast nucleosomes having different fluorescently tagged histones for these studies. Replication occurs in nuclear foci, having “replication factories” with many DNA replication forks. Our recent biochemical and structural studies have defined the composition and atomic structure of the most basic unit of a replication factory, a dimeric replisome. We will employ super resolution microscopy to validate if our reconstituted factory is the same as that inside cells. We have insight into how duplex DNA at origins is opened into single strands from our recent finding that the twin CMG helicases encircling duplex DNA at an origin are directed inward, opposite the “outward” direction thought for a decade. We find that two inward directed CMG can shear DNA apart. We have plans to further this line of investigation.