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

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携带甲氧西林耐药性；(2)了解自负载型启动子的作用机制 解旋酶，使用由SCC元件及其同系物编码的解旋酶作为模型系统 不同系列的移动元件，即SAPI。我们将结合生物化学和生物化学来解决这些问题 结构工具，并与何塞·佩纳德·S博士合作，研究微生物遗传学。 尽管鳞状细胞癌成分与医学相关，但人们对它们的分子生物学知之甚少。 除了将它们整合和切除到宿主染色体内/外的位点特异性重组酶：不 其他核心的“管家”基因已经被定性。通过研究大量的SCC元素，我们 在重组酶周围定义了两种保守的ORF模式。我们对它们序列的分析 表明它们是新的复制模块。 这两种模式都包括复制启动子(Rep)蛋白中具有同源的假定解旋酶 葡萄球菌致病岛(SaPI)。SaPI是一个原本不相关的移动设备家族 比干细胞具有更好的特性，并且已知在切除后可以复制的遗传元件。这个 研究最多的SAPI Rep，即SaPIBov1，是一种自我加载的解旋酶：它识别并打开一个 DsDNA的复制来源，并具有依赖于ATP的解旋酶活性。我们发现SaPIBov1代表 SCCmec IV型同系物是一种活性解旋酶，并确定了它的晶体结构。令人惊讶的是， 与其ATPase结构域最接近的同源物是MCM，古生物/真核复制型解旋酶。 因为这些Rep蛋白很容易使用，所以它们是询问如何自我加载的极佳系统 解旋酶从结合dsDNA转变为围绕单链形成环，并为了理解 并对MCM型AAA+解旋酶的作用机制进行了探讨。 Aim 1 Ask是鳞状细胞癌元件的假定复制蛋白，其功能和确切功能是什么 真的吗？初步结果表明，除了可能的启动子解旋酶外，还包括新的SSB 和一个极简主义的Pola家族聚合酶，这可能是一个底物酶。我们将继续使用生化工具来 计算出它们的体外活性和相互作用。我们的合作者佩纳德·S博士将测试它们提出的功能 在金黄色葡萄球菌体内。(不要求为佩纳德·S博士提供资金)。 目标2问自负载启动子解旋酶是如何工作的？我们将利用我们现有的晶体结构 结合电子显微镜了解这些酶如何与解旋酶中的单链DNA相互作用 模式。为了了解气泡打开的过程，我们将把我们现有的结构与DNA结合起来 足迹、DNA拓扑、其他生物化学和电子显微镜来模拟我们提出的络合物 是两个六角体与~300bp的DNA结合，在泡泡形成前后。

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.