Structural Biology of a Serine DNA Recombinase

这个项目使用x射线晶体学和生物化学的结合来研究基本的 DMA重组中的机械问题。将要详细研究的例子是Sin重组酶， 其由多抗性质粒p！9789金黄色葡萄球菌，一种机会性 感染.与许多相关的质粒编码重组酶一样，Sin似乎促进稳定的质粒 通过解析复制过程中可能形成的二聚体来维持。 Sin是基于丝氨酸的位点特异性DNA重组酶家族的成员。这些酶 广泛存在于原核生物中，进行各种各样的遗传操作，并且越来越受欢迎。 作为基因工程的工具。然而，它们的机制并不像另一种机制那样清楚 位点特异性重组酶的主要家族，基于酪氨酸的家族。 许多位点特异性重组酶仅作为调节其活性的更大复合物的一部分才具有活性 （称为“突触体”，因为它将两个DMA伙伴聚集在一起）。在Sin案例中， 包括另外的Sin四聚体和两种异二聚体 DNA弯曲蛋白该复合物捕获3个双链DNA交叉点，并且只有当 重组位点被适当地定向。然而，这种复合物究竟是如何激活 重组酶未知。丝氨酸重组酶的许多其他机制细节也仍然很差 明白 这项工作的一个主要目标是提供一个结构框架，了解罪恶介导的DNA 重组-一种理解，应广泛适用于大家庭的其他成员， 丝氨酸重组酶。我们的目标是确定整个突触体的结构以及 的各个组成部分。使用化学修饰的寡核苷酸的生物化学实验将 补充这项工作，解决动力学问题，不容易获得的结晶。 这项工作将提高我们的理解，在详细的分子水平上，一种常见的DNA类型 细菌繁殖。这将增强序列数据库的预测能力，并有望 涉及防止细菌中抗性基因的维持和移动的方法。这也将有助于 开发更通用的遗传工具。

This project uses a combination of x-ray crystallography and biochemistry to investigate basic mechanistic questions in DMA recombination. The example that will be studied in detail is Sin recombinase, which is encoded by the multiresistance plasmid p!9789 of Staphylococcus aureus, a cause of opportunistic infections. Like many related plasmid-encoded recombinases, Sin appears to promote stable plasmid maintenance by resolving dimers that can form during replication. Sin is a member of the serine-based family of site-specific DMA recombinases. These enzymes are widespread among prokaryotes, perform a large variety of genetic manipulations, and are becoming popular as genetic engineering tools. However, their mechanism is not nearly as well understood as that of the other major family of site-specific recombinases, the tyrosine-based family. Many site-specific recombinases are active only as part of a larger complex that regulates their activity (termed a "syanptosome", since it brings together the two DMA partners). In the Sin case, the complex includes, in addition to a catalytically active Sin tetramer, an additional Sin tetramer and two heterodimers of the DNA bending protein. The complex entraps 3 double-stranded DNA crossings and can only form if the recombination sites are appropriately oriented. However, exactly how this complex activates the recombinase is not known. Many other mechanistic details of serine recombinases also remain poorly understood. A major goal of this work is to provide a structural framework for understanding Sin-mediated DNA reocombination - an understanding that should be broadly applicable to other members of the large family of serine recombinases. We aim to determine the structure of the entire synaptosome as well as structures of the individual components. Biochemical experiments using chemically modified oligonucleotides will complement this work by addressing kinetic questions not readily accessible by crystallography. This work will improve our understanding, at a detailed molecular level, of a common type of DNA rearrangment in bacteria. This will enhance the predictive power of sequence databases and hopefully lead to ways to prevent the maintenance and mobility of resistence genes in bacteria. It will also help in the development of more versatile genetic tools.