The mechanism(s) used to control the timing and frequency of replication initiation in Gram positive bacteria are unknown. YabA is a negative regulator of replication initiation in B. subtilis, and is conserved in other Gram positive bacteria. The mechanism by which YabA carries out its regulatory function is not yet understood. I found that YabA associates with the chromosomal origin of replication (oriC), and is delivered there at a step following the loading of the replicative helicase. I propose a series of experiments to identify the factor(s) necessary for association of YabA with oriC. I also propose experiments to determine if the subcellular location of YabA and its association with the chromosome changes during the replication cycle. These experiments should provide insights into the timing of YabA's function in inhibition of replication initiation. Finally, I propose several in vivo and in vitro experiments to determine the mechanism of inhibition of replication initiation by YabA. PUBLIC HEALTH RELEVANCE: Regulation of DNA replication is critical for survival and growth, and is generally achieved by controlling the timing and frequency of replication initiation. The mechanisms behind this kind of control remain elusive in a large subset of bacteria known as Gram-positives, a subgroup of which contribute to pathogenesis and human disease. The goal of this project is to study one of the mechanisms behind DNA replication control, and the results of this proposal could contribute to better understanding of these processes, as well as provide general insights into replication control in pathogenic bacteria.