Studies with the human Cdc45-Mcm2-7-GINS helicase complex

描述（由申请人提供）： 研究将使用人类复制解旋酶（由Cdc 45-Mcm 2 -7-GINS（CMG）组成）和复制DNA聚合酶进行，以催化前导链和滞后链的合成。我们的目标是概括在体外的体内观察表明，Pol <$催化前导链DNA合成，而Pol <$plus Pol <$-引发酶复合物支持滞后链合成。我们将利用仅含有三个核苷酸的引发的200-nt环，因此可以使用dATP掺入方便地测量前导链，而滞后链可以随后进行dTTP掺入。我们认为CMG复合物是复制叉的蛋白质核心。参与复制的蛋白质，包括DNA聚合酶、Ctf 4、FACT、Tim-Tipin等，与CMG复合物相互作用并构成移动的复制体。我们计划在体外和体内研究这些相互作用及其对CMG解旋酶活性的影响。CMG复合物组分的改变在DNA复制中起重要作用。已检测到Psf 1（GINS组分）中的特定人类突变，并将研究其对GINS和CMG形成的影响。许多这些突变导致发育缺陷。我们计划确定从Psf 1人类突变体中分离的细胞中的DNA复制水平是否有助于表型缺陷。1

DESCRIPTION (provided by applicant): Project Summary Studies will be carried out with the human replicative helicase, consisting of Cdc45-Mcm2-7-GINS (CMG), and the replicative DNA polymerases in order to catalyze leading and lagging strand synthesis. Our goal is to recapitulate in vitro the in vivo observations suggesting that Pol ¿ catalyzes leading strand DNA synthesis while Pol ¿ plus Pol ¿-primase complex support lagging strand synthesis. We will utilize a primed 200-nt circle containing only three nucleotides so leading strands can be measured conveniently using dATP incorporation while lagging strand can be followed by dTTP incorporation. We propose that the CMG complex is the protein core at the replication fork. Protein involved in replication, including DNA polymerases, Ctf4, FACT, Tim-Tipin, etc. interact with the CMG complex and constitute the moving replisome. We plan to investigate these interactions and their influence on the CMG helicase activity both in vitro and in vivo. Alterations in components of the CMG complex play important roles in DNA replication. Specific human mutations in Psf1 (a GINS component) have been detected and their effects on the formation of GINS and CMG will be investigated. A number of these mutations lead to developmental defects. We plan to determine whether the level of DNA replication in cells isolated from the Psf1 human mutants contribute to phenotypic defects. 1