The bacterial histone-like protein HU has been primarily characterized in Escherichia coli. In addition to its role in genomic DNA compaction, E. coli HU has also been shown to participate in processes such as DNA repair and recombination. However, the range of DNA-binding properties ascribed to HU orthologs in vitro suggest that they serve distinct roles in vivo. A comparative approach to identifying molecular determinants of substrate specificity is implemented. The hypothesis is that the two kinks imposed by HU proteins are 'hinges' that must be secured, and that preference for distorted DNA occurs only if stabilizing contacts are insufficient to secure the hinges in perfect duplex DNA. Site-directed mutagenesis and electrophoretic techniques will be used to measure binding affinity, substrate specificity and site size. Proteins will include HU from Bacillus subtilis and from the hyperthermophile Thermotoga maritima. The hypothesis will also be considered that wrapping of DNA about the surface of HU proteins requires disruption of surface salt bridges. This hypothesis will be evaluated experimentally using the HU homolog TF1, a sequence-specific homolog encoded by a Bacillus bacteriophage, and HU from T. maritima by systematically modifying charged surface residues and measuring binding constants and their salt-dependence. This program focuses on integrating research and teaching by offering the opportunity for both graduate and undergraduate students to acquire research experience and proficiency in techniques associated with analysis of protein-nucleic acid interactions. Graduate students will be integral to developing these research projects, and minority undergraduate students will continue to contribute to related projects.
