Mobile DNA elements function to control variable gene expression and are a major contributor to horizontal gene transfer, including the dissemination of antibiotic resistance and virulence factors. They are also being exploited as potent tools for genetic engineering. Over the years this grant has supported work on the mechanism and control of site-specific DNA recombination reactions, focusing largely on the DNA inversion reaction that regulates flagellar phase variation in Salmonella enterica. We now have a fairly detailed molecular and structural understanding of this reaction, including how the Fis/enhancer regulatory element interacts with the Hin serine recombinase to promote remodeling of Hin dimers into the active synaptic tetramer and how DNA exchange by subunit rotation within the tetramer occurs. Future studies will address two remaining important mechanistic issues: why does the Hin subunit rotation reaction pause at the ligation-competent conformer and how do Fis/enhancer-Hin contacts promote remodeling to the active Hin tetramer. Our emphasis in the next funding period will shift towards understudied recombination reactions mediated by members of the Large Serine Recombinase subfamily, which contain a large C-terminal DNA binding and regulatory domain (CTD), and the IS607-serine transposon subfamily, which have their DNA binding domain at the N-terminus. Regarding the former, we are investigating the Listeria phage A118 serine integrase and its regulatory partner Gp44. Research will be directed at mechanisms underlying the directional control of synapsis and will be influenced by a recently determined X-ray structure of the CTD of a nearly identical Listeria phage integrase by the Van Duyne lab. Regarding serine transposons, our early biochemical and structural studies provide encouragement that we will be able to decipher the novel pathway for transposition by these unusual elements. Since the discovery of the Fis nucleoid protein from our work on the Hin-catalyzed DNA inversion reaction, we have been investigating its many other regulatory roles, its expression as a function of cell growth, and how it selects and deforms it DNA binding site. Under fast growth rates Fis can be the most abundant DNA binding protein in the cell. Fis binds prolifically throughout the chromosome, and we propose is contributing to chromosome compaction through formation of dynamic DNA loops and by DNA bending. In future work we will investigate the role of DNA minor groove shape and Fis-Fis interactions in binding to the chromosome and probe for DNA loops between Fis binding tracts in vivo. We will also investigate cooperative recruitment of proteins by Fis that are primarily mediated through changes in DNA shape.
Work on this project will elucidate mechanisms of inversion, deletion, integration, and complete transposition of DNA segments by enzymes of the serine recombinase family. Knowledge gained from these studies will help to exploit these elements as tools of genetic engineering. An additional area concerns bacterial multi-functional chromatin proteins, particularly regarding their effects on DNA structure, gene regulation, and chromosome packaging.
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