Mechanisms and regulation of serine resolvases

描述（由申请人提供）：这项工作的长期目标是在分子细节上了解丝氨酸重组酶的调控和催化机制。这些位点特异性DNA重组酶在原核生物中广泛存在，并执行各种遗传操作，通常与抗性基因的传播和/或稳定性有关。它们也是操纵其他生物体基因组的有用工具。然而，它们并不像另一个位点特异性重组酶家族，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分解亚家族，以Sin为原型。Sin由葡萄球菌的许多大的多抗性质粒编码。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控作用：WT酶催化的是分子内重组而不是分子间重组，可以感知其位点的相对方向，尽管没有化学键能的净释放，但可以定向交换链。这种调节的关键在于，它们只在一个被称为“突触体”的大而交织的复合体中活跃。因为底物拓扑结构极大地促进（或者，在其他情况下，抑制）突触体的形成，它就像一个“拓扑过滤器”。在确定的突触体拓扑结构内，链交换释放超卷曲张力，为偏向反应方向提供能量来源。调控复合体如何激活二聚体结合到配对的交叉位点是未知的。这些酶用于催化DNA链断裂和团聚的机制也知之甚少。已知它们形成磷酸丝氨酸中间体，但迄今为止可用的结构并未显示出完全组装的活性位点，这使得难以解释保守残基的化学作用。了解丝氨酸分解酶的突触复合体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调节装置的丝氨酸重组酶，其从非活性到活性转变的分子细节也可能是类似的。本研究采用的方法是体外生化研究和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。这项工作探讨了DNA重组反应的机制和调控，这对于在金黄色葡萄球菌中发现的许多耐药质粒的稳定维持是重要的，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/或迁移。这项工作将有助于更好地了解细菌的遗传交易，提高序列数据库的预测能力，并为控制细菌中的抗性基因提供途径。

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.