Lifestyle of the SCCmec element and mechanisms of self-loading helicases

SCCmec 元件的生活方式和自加载解旋酶的机制

• 批准号: 9923690
• 负责人: PHOEBE A RICE
• 金额: $ 44.04万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9923690
• 关键词: ATP Hydrolysis; ATPase Domain; Address; Appearance; Basic Science; Binding; Biochemical; Biochemistry; Bioinformatics; Biological Assay; Biological Models; Chromosomes; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; DNA; DNA Binding; DNA Footprint; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; Electron Microscopy; Elements; Enzymes; Epidemic; Event; Excision; Experimental Designs; Family; Funding; Future; Genome; Genus staphylococcus; Goals; Homologous Gene; Housekeeping Gene; In Vitro; Label; Life Style; Maps; Medical; Methicillin Resistance; Microbial Genetics; Mobile Genetic Elements; Modeling; Molecular Biology; Molecular Conformation; Negative Staining; Nucleotides; Open Reading Frames; Pathogenicity Island; Pattern; Plasmids; Polymerase; Process; Proteins; RNA; Replication Origin; Resolution; SS DNA BP; Site; Staphylococcus aureus; Structure; System; Testing; Torque; Toxic Shock Syndrome; Toxin; United States National Institutes of Health; Virulence Factors; Work; analog; biochemical tools; cofactor; ds-DNA; expression cloning; helicase; in vitro activity; in vivo; methicillin resistant Staphylococcus aureus; novel; recombinase; tool

Project Summary/Abstract: This work has two overall goals: (1) to understand the lifestyle of the mobile genetic elements (SCCs) that carry methicillin resistance in S. aureus, and (2) to understand the mechanism of self-loading initiator helicases, using as model systems ones that are encoded by SCC elements and their homologs from a different family of mobile elements, the SaPIs. We will address these with a combination of biochemical and structural tools, and in collaboration with Dr. José Penadés, microbial genetics. Despite the medical relevance of SCC elements, very little is known about their molecular biology beyond the site-specific recombinases that integrate and excise them into / out of the host chromosome: no other core “housekeeping” genes have been characterized. By examining numerous SCC elements, we defined two patterns of conserved ORFs surrounding the recombinases. Our analysis of their sequences suggests that they are novel replication modules. Both patterns include a putative helicase with a homolog among the replication initiator (“Rep”) proteins of the staphylococcal pathogenicity islands (SaPIs). The SaPIs are an otherwise-unrelated family of mobile genetic elements that are better characterized than the SCCs and are known to replicate after excision. The best-studied SaPI Rep, that of SaPIBov1, is a self-loading helicase: it recognizes and opens a bubble in an origin of replication in dsDNA, and has ATP-dependent helicase activity. We found that the SaPIBov1 Rep homolog from SCCmec type IV is an active helicase and determined its crystal structure. Surprisingly, the closest structural homolog to its ATPase domain is MCM, the archaeal / eukaryotic replicative helicase. Because these Rep proteins are easy to work with, they are excellent systems for asking how self-loading helicases morph from binding dsDNA to forming a ring around a single strand, and for understanding the mechanism of MCM-type AAA+ helicases as well. Aim 1 asks are the putative replication proteins of SCC elements functional and what exactly do they do? Preliminary results suggest that as well as the putative initiator helicases, these include novel SSBs and a minimalist PolA family polymerase that may be a primase. We will continue use biochemical tools to work out their in vitro activities and interactions. Our collaborator Dr. Penadés will test their proposed functions in vivo in S. aureus. (No funds are requested for Dr. Penadés). Aim 2 asks How do self-loading initiator helicases work? We will use our existing crystal structure in conjunction with electron microscopy to understand how these enzymes interact with ssDNA in helicase mode. To understand the process of bubble opening, we will combine our existing structure with DNA footprinting, DNA topology, other biochemistry and electron microscopy to model the complex that we propose is two hexamers bound to ~300bp of DNA, before and after bubble formation.

项目摘要/摘要： 这项工作有两个总体目标：（1）了解移动遗传元素（SCC）的生活方式 在金黄色葡萄球菌中具有甲氧西林的耐药性，（2）了解自动加载引发剂的机制 解旋酶，使用作为模型系统，由SCC元素及其同源物编码的模型系统。 移动元素的不同家族，SAPI。我们将结合生化和 结构工具，并与微生物遗传学的JoséPenadés博士合作。 尽管SCC元素的医学相关性，但对它们的分子生物学知之甚少 除了将其整合并将其锻炼到宿主染色体的特定地点重物组织酶之外：否 其他核心“家政”基因也被描述了。通过检查许多SCC元素，我们 定义了重物组织酶周围保守的ORF的两种模式。我们对它们序列的分析 表明它们是新颖的复制模块。 这两种模式都包括一个假定的解旋酶，在复制引发剂（“ REP”）蛋白中具有同源物 葡萄球菌致病岛（SAPI）的SAPI是一个原本与手机的无关家族 比SCC更好地表征遗传元素，并在惊喜后复制。这 Sapibov1的最佳研究SAPI代表是一个自载的解旋酶：它识别并打开了一个气泡 DSDNA中复制的起源，并具有ATP依赖性解旋酶活性。我们发现Sapibov1代表 来自SCCMEC IV类的同源物是一种活性解旋酶，并确定其晶体结构。令人惊讶的是， 最接近其ATPase结构域的结构同源物是MCM，MCM，档案 /真核复制型解旋酶。 因为这些代表蛋白易于使用，所以它们是询问自我载荷的绝佳系统 解旋酶从结合dsDNA变为围绕单链形成一个环，并理解 MCM型AAA+解旋酶的机理。 AIM 1问是SCC元素功能的推定复制蛋白 他们做吗？初步结果表明，除了推定的引发剂解旋酶，其中包括新型SSB 以及一种极简主义的Pola家族聚合酶，可能是一种原始酶。我们将继续使用生化工具来 确定他们的体外活动和互动。我们的合作者Penadés博士将测试他们建议的功能 体内的金黄色葡萄球菌。 （没有要求Penadés博士的资金）。 AIM 2询问自加载引发剂解放酶如何起作用？我们将使用现有的晶体结构 与电子显微镜结合使用，以了解这些酶如何与解旋酶中的ssDNA相互作用 模式。为了了解泡沫打开的过程，我们将现有的结构与DNA相结合 足迹，DNA拓扑，其他生物化学和电子显微镜，以对我们提出的复合物进行建模 是在气泡形成之前和之后的两个六聚体约为300bp的DNA。