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.

R56GM066011

2002-08-15

2009-07-31