Mechanisms and regulation of serine resolvases

描述（由申请人提供）：这项工作的长期目标是从分子上详细了解丝氨酸重组酶的调控和催化机制。这些位点特异性DNA重组酶广泛存在于原核生物中，并进行各种各样的遗传操作，通常与抗性基因的传播和/或稳定性相关。它们也是操纵其他生物基因组的有用工具。然而，他们并不像其他家庭的位点特异性重组酶，酪氨酸或？整合酶家族（包括Cre和Flp）。这项工作将集中在丝氨酸重组酶的resolvase亚家族，使用Sin作为原型。Sin由葡萄球菌的许多大的多重抗性质粒编码。金黄色葡萄球菌，并且被认为通过将复制子二聚体分解成单体来促进它们的稳定维持。丝氨酸分解酶的一个显著特征是它们的调节：WT酶将催化分子内而不是分子间的重组，可以感知它们位点的相对方向，并且可以定向交换链，尽管没有化学键能的净释放。这种调节的关键是它们只在一个叫做“突触体”的大的、相互缠绕的复合体中活跃。因为基底拓扑结构极大地促进（或在其他情况下，抑制）突触体的形成，所以它起着“拓扑过滤器”的作用。“在突触体的定义拓扑结构内，链交换释放超螺旋张力，提供能量来源来偏置反应方向。调控复合物如何激活结合到配对交叉位点的二聚体尚不清楚。这些酶催化DNA链断裂和重新结合的机制也知之甚少。已知它们形成磷酸丝氨酸中间体，但迄今为止可用的结构没有显示出完全组装的活性位点，使得难以解释保守残基的化学作用。了解丝氨酸解离酶的突触复合物如何调节重组的化学和机械步骤应该适用于其他丝氨酸重组酶：即使对于那些需要不同的调节装置的丝氨酸重组酶，无活性到活性过渡的分子细节也可能是类似的。本研究采用的方法是体外生物化学研究和X射线晶体学的结合，与具有遗传学，生物化学和DNA拓扑学专业知识的小组密切合作。公共卫生相关性这项工作探讨了DNA重组反应的机制和调控，该反应对于稳定维持金黄色葡萄球菌中发现的许多携带耐药性的质粒至关重要，金黄色葡萄球菌是机会性感染的常见原因。在其他细菌中发现的密切相关的重组系统也有助于抗性基因的维持和/或移动。这项工作将导致更好地了解细菌中的遗传交易，增强序列数据库的预测能力，并导致控制细菌中抗性基因的方法。

DESCRIPTION (provided by applicant): The long-term goal of this work is to understand, in molecular detail, the regulation and catalytic mechanism of serine recombinases. These site-specific DNA recombinases are widespread in prokaryotes and perform a wide variety of genetic manipulations, often associated with the spread and/or stability of resistance genes. They are also useful tools for manipulating the genomes of other organisms. However, they are not as well understood as the other family of site-specific recombinases, the tyrosine or ? integrase family (which includes Cre and Flp). This work will focus on the resolvase subfamily of serine recombinases, using Sin as a prototype. Sin is encoded by many large multi-resistance plasmids of Staph. aureus, and is thought to promote their stable maintenance by resolving replicon dimers into monomers. A remarkable feature of the Serine resolvases is their regulation: the WT enzymes will catalyze intrabut not intermolecular recombination, can sense the relative orientation of their sites, and can exchange strands directionally despite the fact that there is no net release of chemical bond energy. This key to this regulation is that they are only active within a large, intertwined complex called the "synaptosome." Because substrate topology greatly facilitates (or, in other cases, inhibits) formation of the synaptosome, it acts as a "topological filter." Within the defined topology of the synaptosome, strand exchange releases supercoiling tension, providing an energy source to bias the reaction direction. How the regulatory complex activates the dimers bound to the paired crossover sites is unknown. The mechanism used by these enzymes to catalyze the breakage and reunion of DNA strands is also poorly understood. It is known that they form phosphoserine intermediates, but the structures available to date do not show a fully assembled active site, making it difficult to interpret the chemical roles of conserved residues. Understanding how the synaptic complexes of the serine resolvases regulate the chemical and mechanical steps of recombination should be applicable to other serine recombinases: even for those that require a different regulatory apparatus, the molecular details of the inactive-to-active transition is likely to be analogous. The approach taken in this study is a combination of in vitro biochemical studies and x-ray crystallography, in close collaboration with a group that has expertise with genetics, biochemistry, and DNA topology. PUBLIC HEALTH RELEVANCE This work probes the mechanism and regulation of a DNA recombination reaction that is important for stable maintenance of many drug resistance-carrying plasmids found in Staphylococcus aureus, a common cause of opportunistic infections. Closely related recombination systems found in other bacteria also aid in the maintenance and/or mobility of resistance genes. This work will lead to a better understanding of genetic transactions in bacteria, enhancing the predictive power of sequence databases and leading toward ways to control resistance genes in bacteria.