Evolution of Function of Bacterial Nucleoid-associated Proteins

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The focus of this program is on the organization of the bacterial nucleoid. The bacterial genome is associated with a number of DNA-binding proteins. Some, such as HU proteins, are ubiquitous, while others are only encoded by a subset of eubacteria. Dps (DNA protection during starvation) proteins belong to the latter category. A main goal is to define molecular determinants of function within these protein families. The focus will be on the radiation-resistant eubacterium Deinococcus radiodurans which encodes one HU and two Dps homologs. For D. radiodurans HU, its spatial disposition on its preferred substrate, the four-way DNA junction, will be determined using site-directed mutagenesis and photochemical crosslinking. Roles in nucleoid organization will be assessed by in vivo localization and analysis of expression levels during normal exponential growth as well as in response to DNA damage. For Dps-1, the mode of DNA interaction will be determined in vitro and its expression as a function of growth condition or environmental stress will be determined. As Dps-2 is predicted to be non-cytoplasmic, its cellular localization will be determined, along with its mode of DNA binding and regulation in response to stress. It is anticipated that the proposed analyses will contribute not only to our mechanistic understanding of DNA binding by members of two important families of nucleoid-associated proteins, but also to elucidating cellular processes that contribute to the exceptional resistance to environmental stress that is characteristic of D. radiodurans. This program focuses on integrating research and teaching by offering the opportunity for both graduate and undergraduate students to acquire research experience. The involvement of minority students will be emphasized, and students at all levels will be expected to disseminate their research at national meetings.