STRUCTURE DETERMINATION OF PROTEINS INVOLVED IN DNA REPLICATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 一个正在进行的项目，旨在确认，与MALDI-MS，识别各种蛋白质参与DNA复制。 这些蛋白质被过度表达和重组，用于X射线晶体学测定其三维结构。 S.酿酒酵母和B. subtilus和S.酿酒酵母是正在研究的蛋白质复合物之一。 微染色体维持蛋白（MCM）在古生菌和真核生物中作为复制解旋酶。有趣的是，通过BLAST分析，在细菌蜡状芽孢杆菌（Bc）中整合的噬菌体内鉴定了MCM同源物。BcMCM仅与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取代。我们发现BcMCM显示3 '->5'解旋酶和ssDNA刺激的ATP酶活性，这些特性来自其保守的AAA结构域。分离的BcMCM在溶液中是单体，但可能在体内形成功能性寡聚体。我们发现，BcMCM氨基端可以结合ssDNA和窝藏一个锌原子，这两个标志的典型MCM氨基端。未检测到BcMCM催化的引发酶活性。我们认为BcMCM的发散氨基端是MCM解旋酶相应区域的一个部分。发散的氨基末端使BcMCM成为典型MCM-解旋酶的有用模型，因为它使用明显不相关的结构实现相同的功能。这项工作已发表在《核酸研究》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.