DNA REPLICATION PROTEIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在真核生物中，DNA复制的启动需要一组称为起源识别复合体(Origin Recognition Complex，ORC)的蛋白质，它与DNA上称为复制起点的特定位置结合。最近几个实验室的工作(有些尚未发表)确定了酵母和果蝇兽人的低分辨率结构，但这些都是有争议的，因为尽管ORC蛋白是同源的，但EM结构明显不同。古菌利用的过程比细菌更类似于真核生物的过程，但组织要简单得多，因此为帮助理解真核过程提供了一个很好的模型系统。我们是唯一能够在体外组装古生菌复制起点的实验室(手稿正在审查中)。我们可以将8个相同的ORC1蛋白亚基与350bp的起始DNA(总MR~600 KDa)结合。一旦所有八个ORC1亚基都与起始点结合，该复合体就会启动起始点内富含AT的区域的解离，作为加载复制解旋酶的前奏。我们已经有了ORC1蛋白质单体和二聚体的晶体结构，但我们想了解当功能起始点被组装时亚基之间的相互作用，以及这种组装如何导致复制起始点的解开。虽然我们正在努力使整个复合体结晶，但这是一个雄心勃勃的目标，在没有晶体的情况下，EM结构将对了解组装过程有很大帮助，特别是在复制起始复合体中N-末端AAA+结构域如何相互作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Initiation of DNA replication in eukaryotes requires a group of proteins called the Origin Recognition Complex (ORC) which binds to specific sites on DNA called replication origins. Recent work in several labs (some as yet unpublished) has determined the low resolution structures of the ORCs of Yeast and Drosophila, but these are controversial because, although the ORC proteins are homologous, the EM structures are significantly different. Archaea utilise a process more similar to that seen in eukaryotes than bacteria but the organisation is much more simple and hence provides a good model system in helping the understand the eukaryotic process. We are the only lab to be able to assemble an archaeal replication origin in vitro (manuscript under review). We can bind 8 identical ORC1 protein subunits to the 350bp origin DNA (total Mr ~600KDa). Once all eight ORC1 subunits have bound to the origin, the complex then initiates unwinding of an AT-rich region within the origin as a prelude to loading the replicative helicase. We already have crystal structures of the monomeric and dimeric forms of the ORC1 protein alone but we would like to understand the interactions between the subunits when a functional origin is assembled and how this assembly induces unwinding of the replication origin. Although we are trying to crystallise the entire complex, this is an ambitious goal and in the absence of crystals an EM structure would be of enormous help in understanding the assembly process, in particular how the N-terminal AAA+ domains interact with each other within the replication origin complex.