STRUCTURE DETERMINATION OF PROTEINS INVOLVED IN DNA REPLICATION

DNA 复制涉及的蛋白质的结构测定

• 批准号: 8361535
• 负责人: David Jeruzalmi
• 金额: $ 0.13万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361535
• 关键词: ATP phosphohydrolase; Archaea; Bacillus cereus; Bacteria; Bacteriophages; Binding; Chromosomes; DNA Primase; DNA biosynthesis; Eukaryota; Funding; Grant; Homologous Gene; Maintenance; Modeling; National Center for Research Resources; Nucleic Acids; Principal Investigator; Property; Protein S; Proteins; Publishing; Research; Research Infrastructure; Resources; Roentgen Rays; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; United States National Institutes of Health; Work; Zinc; cost; helicase; in vivo; macromolecule; monomer; origin recognition complex; overexpression; paralogous gene; protein complex; reconstitution; three dimensional structure

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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: A biochemically active MCM-like helicase in Bacillus cereus. Samuels M, Gulati G, Shin JH, Opara R, McSweeney E, Sekedat M, Long S, Kelman Z, Jeruzalmi D. Nucleic Acids Res. 2009 Jul;37(13):4441-52

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 一个正在进行的项目旨在利用 MALDI-MS 确认 DNA 复制中涉及的各种蛋白质的鉴定。 这些蛋白质被过度表达和重组，目的是通过 X 射线晶体学测定其三维结构。 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 蛋白属于正在研究的蛋白质复合物。 微型染色体维持（MCM）蛋白在古细菌和真核生物中充当复制解旋酶。有趣的是，通过 BLAST 分析，在蜡样芽孢杆菌 (Bc) 细菌中整合的噬菌体中鉴定出 MCM 同源物。 BcMCM 仅与 MCM 解旋酶的 AAA 区域相关；典型的氨基末端缺失，并被与引物酶同源性较弱的片段取代。我们表明，BcMCM 显示出 3'-->5' 解旋酶和 ssDNA 刺激的 ATP 酶活性，这些特性源自其保守的 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低聚物。我们发现 BcMCM 氨基末端可以结合 ssDNA 并含有锌原子，这都是典型 MCM 氨基末端的标志。未检测到 BcMCM 催化的引物酶活性。我们认为 BcMCM 的不同氨基末端是 MCM 解旋酶相应区域的旁系同源物。不同的氨基末端使 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完成相同的功能。该作品已发表： 蜡样芽孢杆菌中具有生化活性的 MCM 样解旋酶。 Samuels M、Gulati G、Shin JH、Opara R、McSweeney E、Sekedat M、Long S、Kelman Z、Jeruzalmi D。核酸研究。 2009年7月；37(13):4441-52