STRUCTURE DETERMINATION OF PROTEINS INVOLVED IN DNA REPLICATION

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。 一个正在进行的项目，旨在用MALDI-MS确认与DNA复制有关的各种蛋白质的鉴定。这些蛋白质被过度表达和重组，用于X射线结晶学测定其三维结构。来自酿酒酵母和枯草芽孢杆菌的MCM蛋白，以及来自酿酒酵母的ORC蛋白是正在研究的蛋白质复合体。 微小染色体维持(MCM)蛋白是古生物和真核生物中的复制型解旋酶。有趣的是，通过BLAST分析，在整合到蜡状芽孢杆菌(BC)中的噬菌体中鉴定出一个MCM同源物。BcMCM只与MCM解旋酶的AAA区相关；典型的氨基末端缺失，并被与引物酶具有弱同源性的片段所取代。我们发现BcMCM显示了3‘-&gt；5’解旋酶和ssDNA刺激的ATPase活性，这一特性源于其保守的AAA结构域。分离的BcMCM是溶液中的单体，但在体内可能形成功能性低聚物。我们发现BcMCM氨基端可以与单链DNA结合，并含有一个锌原子，这两个特征都是典型的MCM氨基端的特征。未检测到BcMCM催化的底物酶活性。我们认为，BcMCM的发散氨基末端是MCM解旋酶相应区域的平行对数。发散的氨基末端使BcMCM成为典型的MCM解旋酶的有用模型，因为它使用一个明显无关的结构来完成相同的功能。这项研究已发表在《核酸研究》杂志上。

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. An ongoing project aimed at confirming, with MALDI-MS, the identification of various proteins involved in DNA replication. These proteins are being overexpressed and reconstituted for the purpose of X-Ray crystallographic determination of their three-dimensional structures. MCM proteins from S. cerevisiae and B. subtilus, and ORC proteins from S. cerevisiae are among the protein complexes under study. The mini-chromosome maintenance (MCM) proteins serve as the replicative helicases in archaea and eukaryotes. Interestingly, an MCM homolog was identified, by BLAST analysis, within a phage integrated in the bacterium Bacillus cereus (Bc). BcMCM is only related to the AAA region of MCM-helicases; the typical amino-terminus is missing and is replaced by a segment with weak homology to primases. We show that BcMCM displays 3'-->5' helicase and ssDNA-stimulated ATPase activity, properties that arise from its conserved AAA domain. Isolated BcMCM is a monomer in solution but likely forms the functional oligomer in vivo. We found that the BcMCM amino-terminus can bind ssDNA and harbors a zinc atom, both hallmarks of the typical MCM amino-terminus. No BcMCM-catalyzed primase activity could be detected. We propose that the divergent amino-terminus of BcMCM is a paralog of the corresponding region of MCM-helicases. A divergent amino terminus makes BcMCM a useful model for typical MCM-helicases since it accomplishes the same function using an apparently unrelated structure. This work has been published in Nucleic Acids Research.