Mechanisms and regulation of serine resolvases

描述(申请人提供)：这项工作的长期目标是从分子细节上了解丝氨酸重组酶的调节和催化机制。这些位点特异的DNA重组酶广泛存在于原核生物中，执行各种各样的遗传操作，通常与抗性基因的传播和/或稳定性有关。它们也是操纵其他生物基因组的有用工具。然而，人们对它们的理解不如另一类位点特异性重组酶，酪氨酸或？整合酶家族(包括Cre和FLP)。本工作将以Sin为原型，重点研究丝氨酸重组酶的分解酶亚家族。SIN由金黄色葡萄球菌的许多大的多抗质粒编码。被认为是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，可以感知其位点的相对方向，并且可以定向交换链，尽管没有净释放化学键能量。这一调控的关键在于，它们只活跃在一个巨大的、相互交织的复合体中，称为“突触体”。因为底物拓扑极大地促进(或，在其他情况下，抑制)突触体的形成，它起到“拓扑过滤器”的作用。在已定义的突触体拓扑结构中，链交换释放超螺旋张力，提供能量来源来偏向反应方向。调控复合体如何激活与配对的交叉位点结合的二聚体尚不清楚。这些酶用来催化DNA链断裂和团聚的机制也鲜为人知。众所周知，它们形成了磷酸丝氨酸中间体，但到目前为止可用的结构还没有显示出完全组装的活性部位，这使得很难解释保守残基的化学作用。理解丝氨酸分解酶的突触复合体如何调控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控装置的重组酶，从无效到活跃转变的分子细节也可能是相似的。这项研究采用的方法是体外生化研究和X射线结晶学的结合，并与一个在遗传学、生物化学和DNA拓扑学方面拥有专业知识的小组密切合作。公共卫生相关性这项工作探索了DNA重组反应的机制和调节，DNA重组反应对于稳定维持在金黄色葡萄球菌中发现的许多携带耐药性的质粒非常重要，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，并导致控制细菌中耐药基因的方法。

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.