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.
Intellectual merit: The bacterium Deinococcus radiodurans is particularly resistant to agents that damage its cellular components, including its genomic DNA. This makes D. radiodurans a good model system for understanding how protection against damage is accomplished and how damage is repaired. In this program the focus has been on proteins that participate in preventing DNA damage resulting from exposure to reactive oxygen species. Reactive oxygen species are produced naturally during aerobic respiration, and they can also be produced from external sources such as ionizing radiation or as part of antibacterial defense mechanisms. It is therefore important to understand how cells respond to the presence of reactive oxygen species and minimize damage to their genomic DNA and the attendant risk of incurring gene mutations or cell death. This program focused on two highly conserved bacterial proteins, HU and Dps, both of which are thought to be associated with the genomic DNA and important for its compaction. D. radiodurans encodes one HU homolog and two Dps homologs, named Dps-1 and Dps-2. Specific outcomes include a demonstration of the mechanism by which Dps-1 interacts with DNA, showing the simultaneous association of Dps-1 with four DNA molecules. Such arrangement would allow for a compact array in which DNA is protected from damaging agents; DNA compaction was confirmed in vivo by visualization of the genomic DNA in cells in which Dps-1 is overexpressed. It was also shown that DNA binding by Dps-1 is sensitive to the presence of reactive oxygen species, further attesting to its role in responding to such conditions. In contrast, overexpression of D. radiodurans HU does not lead to genomic DNA compaction beyond what is seen in wild-type cells, but HU is expressed at higher levels in response to the presence of reactive oxygen species. Overexpression of Dps-2 likewise does not lead to genomic DNA compaction; rather, Dps-2 appears to be localized to the periplasm and not in direct contact with the genomic DNA. Taken together, our data have established the mode of interaction between Dps and DNA; since Dps does not contain a classical DNA-binding motif, this analysis has contributed significantly to understanding the mechanism of Dps-DNA interaction, which will be of general importance to understanding compaction of bacterial genomic DNA. Our data also indicate that efficient protection against externally derived reactive oxygen species is likely afforded through a combination of both compaction of genomic DNA by association with the conserved HU and Dps proteins as well as interception of reactive oxygen species on encounter with scavengers of reactive oxygen species that are localized near the cell membrane. Broader impact: This program has afforded the opportunity for multiple students to participate in research, and it has provided an avenue for the inclusion of members of groups underrepresented in the sciences. Several graduate and undergraduate students have received training in molecular, biochemical, and genetic approaches to understanding protein-DNA interaction. Both graduate and undergraduate students have published their research in peer-reviewed journals, and they have presented their data at national conferences. These experiences have been key to their professional and career development and essential to the training of future scientists. For graduate students, such productive research experiences have enabled them to secure faculty or postdoctoral positions following graduation, while undergraduate students have become more competitive for admission to professional programs.
