Human tuberculosis caused by Mycobacterium tuberculosis is the most prevalent and deadly bacterial infectious disease worldwide. This problem is compounded by the emergence of strains of M. tuberculosis that are resistant to one or more anti-tuberculous drugs. Following initial infections, M. tuberculosis frequently enters a latent or dormant state for extended periods and subsequently, under appropriate conditions or following immune suppression, revives, multiplies and causes a secondary infection. DNA replication constitutes an important step in the exit from latency. The development of novel therapeutic agents to control M. tuberculosis infections in HIV infected patients as well as other individuals is severely hindered by our limited understanding of the initiation and regulation of M. tuberculosis DNA replication and its coordination with other events in cell cycle. Initiation of DNA replication is believed to be triggered when DnaA, the putative initiator protein, interacts with oriC or origin of replication. Although oriC is essential for survival, some clinical strains of M. tuberculosis appear to tolerate major deletions and IS6110 insertions in their oriC, thereby raising questions as to how these clinical strains replicate their genome. Our research proposal focuses on understanding the replication initiation process in M. tuberculosis. Specifically, we propose to inactivate oriC, dnaA individually and together by homologous recombination in an attempt to determine whether replication in M. tuberculosis can proceed from alternate origins, and if so whether dnaA function is required for such replication. The interactions of DnaA with replication origins and consequences of these interactions will be investigated using biochemical and genetic approaches. To begin identifying the factors that could potentially affect DnaA activity, a proteomic approach combining two-dimensional gel electrophoretic separations of proteins with subsequent identification of protein spots by matrix-assisted laser ionization desorption/ionization mass spectrometry, will be used. Defining the molecular events involved in the initiation and regulation of replication is an essential prerequisite for developing defined systems for identifying novel antimycobacterial compounds, and thereby preventing the development of potentially lethal infections of M. tuberculosis.