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

项目概要/摘要： 本工作有两个总体目标：（1）了解移动的遗传元件（SCC）的生活方式 携带甲氧西林耐药性的沙门氏菌。了解自装填引发剂的作用机理 解旋酶，使用由SCC元件编码的解旋酶作为模型系统， 不同系列的移动的元素，即SaPI。我们将结合生物化学和 结构工具，并与José Penadés博士合作，微生物遗传学。 尽管SCC元素具有医学相关性，但对其分子生物学知之甚少 除了将它们整合到宿主染色体中或从宿主染色体中切除的位点特异性重组酶之外：否 其他核心“持家”基因已被鉴定。通过检查众多SCC元素，我们 定义了重组酶周围的两种保守ORF模式。我们对它们序列的分析 表明它们是新的复制模块。 这两种模式都包括一个假定的解旋酶与复制启动子（“Rep”）蛋白之间的同源物 葡萄球菌致病岛（SaPI）SaPI是一个与移动的产品无关的家族 比SCC更好地表征的遗传元件，并且已知在切除后复制。的 研究得最好的SaPI Rep，即SaPIBov 1，是一种自加载解旋酶：它识别并打开一个气泡， dsDNA中的复制起点，并具有ATP依赖性解旋酶活性。我们发现， 来自SCCmec IV型的同系物是活性解旋酶，并确定了其晶体结构。令人惊讶的是， 与其ATP酶结构域最接近的结构同源物是MCM，即古细菌/真核生物复制解旋酶。 因为这些Rep蛋白很容易处理，所以它们是研究自我加载如何 解旋酶从结合dsDNA转变为形成围绕单链的环，并且为了理解解旋酶的结构， MCM型AAA+解旋酶的机制。 目的1是问SCC元件的假定复制蛋白是否有功能，以及确切的功能是什么 是吗初步结果表明，以及推定的起始解旋酶，这些包括新的SSB 和可能是引发酶的极简PolA家族聚合酶。我们将继续使用生化工具， 研究它们的体外活性和相互作用。我们的合作者Penadés博士将测试他们提出的功能 在S.金黄色。(No为Penadés博士申请资金）。 目标2：自装载引发剂解旋酶如何工作？我们将利用现有的晶体结构 结合电子显微镜来了解这些酶如何与解旋酶中的ssDNA相互作用 模式为了理解气泡打开的过程，我们将联合收割机与DNA结合 足迹，DNA拓扑结构，其他生物化学和电子显微镜来模拟我们提出的复合物 是在气泡形成之前和之后与~ 300 bp的DNA结合的两个六聚体。

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.