Unexpected parallels between SaPI replication initiators and conserved SCC ORFs

 描述(申请人提供)：这个项目将调查一个关于携带甲氧西林耐药性的移动遗传元件(SCCmec)如何在金黄色葡萄球菌菌株之间传播的新假说：我们建议它编码以前未知的功能用于自身复制，这将提高任何水平基因转移机制的效率。耐甲氧西林金黄色葡萄球菌(MRSA)是一个严重且日益严重的公共卫生问题。整合和切除SCCmec元件进入和离开宿主基因组的重组酶已经被鉴定出来，并在我们的实验室进行研究。然而，关于从一个基因组中切除和在另一个基因组中出现之间的事件，我们一无所知。在分析重组酶基因两侧保守的ORF时，我们意识到其中一些可能具有DNA复制功能。这一建议集中在我们发现的一个大的假定的ATPase(CCH)，它与金黄色葡萄球菌致病岛(SaPI)编码的已知复制起始蛋白有关。我们的初步结构和生化数据表明，所讨论的SCCmec蛋白(CCH)是一种自负载型解旋酶，其在ATPase结构域中最接近的同源物是真核和古生物复制的MCM解旋酶。因此，我们已经确定了该类型解旋酶在活性环形式中的第一结构。这项工作将改变人们对SCCmec元件的看法以及它们如何传播甲氧西林耐药性，并将为理解自负载和MCM样解旋酶的机制提供新的模型系统。目标1：对CCH的结构理解目标2：SAPI/SCC复制启动子的亚型在结构上有多相似/不同？目的3：CCH和SaPI3rep的生化活性是什么？

 DESCRIPTION (provided by applicant): This project will investigate a new hypothesis regarding how the mobile genetic element (SCCmec) that carries methicillin resistance spreads among S. aureus strains: We propose that it encodes previously unsuspected functionality for its own replication, which would enhance the efficiency of any horizontal gene transfer mechanism. MRSA (methicillin resistant S. aureus) is a serious and growing public health problem. The recombinases that integrate and excise the SCCmec element into and out of the host genome have been identified and are under study in our lab. However, nothing is known about the events between excision from one genome and appearance in another. While analyzing the conserved ORFs that flank recombinase genes, we realized that some of are likely to have functions in DNA replication. This proposal focuses on a large putative ATPase (Cch) that we discovered is related to the known replication initiation proteins encoded by S. aureus pathogenicity islands (SaPIs). Our preliminary structural and biochemical data suggest that the SCCmec protein in question (Cch) is a self-loading helicase whose closest homologs in the ATPase domain are, surprisingly, the eukaryotic and archaeal replicative MCM helicases. Thus we have determined the first structure of that type of helicase in the active ring form. This work will change how people think about SCCmec elements and how they spread methicillin resistance and will provide new model systems for understanding mechanisms of self-loading and MCM-like helicases. Aim 1: Structural understanding of Cch Aim 2: How structurally similar / different are the subtypes of SaPI / SCC replication initiator? Aim 3: What are the biochemical activities of Cch and SaPI3 rep?