M. tuberculosis, the causative agent of tuberculosis is a well known pathogen that has reestablished itself as a significant world wide public health hazard. While there is a great deal of published information in the physiology and pathogenesis of M. tuberculosis, there is very little information available on genetic control of DNA synthesis and related metabolism governing DNA replication. Replication of bacterial chromosomes occurs at unique sequences called 'oriC' and is thought to be regulated at the level of initiation. Studies on replication initiation in other bacteria suggest that both DnaA, the initiator protein and replication origin sequences are structurally conserved. The replication origin is AT rich and contains repeats of nine nucleotide long DnaA protein recognition sequences called the DnaA boxes and repeats of 13 - 16 nucleotide long AT rich elements. The number and position of some of the DnaA boxes is variable in each bacteria. The dnaA gene flanking region in many bacteria supports autonomous replication activity. The genetic and biochemical aspects of replication initiation in mycobacteria are not known. Our research proposal focuses on identifying the key elements needed for replication initiation in M. tuberculosis. During the granting period M. tuberculosis dnaA gene will be cloned and its nucleotide sequence will be determined. The nucleotide sequence of the dnaA flanking region will be determined and putative replication origin sequence features, if any, will be identified. The ability of the dnaA gene flanking sequences to support autonomous replication when present in nonreplicative plasmids will be determined. Sequential deletions from both the 5' and the 3' end of the flanking regions will be carried out to identify the minimal size DNA fragment that supports autonomous replication. If dnaA gene flanking region is not the 'oriC', then shot gun cloning approach will be used to identify the replication origin sequences. Once M. tuberculosis oriC is identified, then oligonucleotide primers specific to oriC will be synthesized and used to amplify related replication origin sequences from the genomic DNA preparations of other species of mycobacteria. The nucleotide sequence of these replication origins will be determined and compared to determine similarities and dissimilarities in the organization of 'oriC' in mycobacteria. M. tuberculosis DnaA protein will be overproduced in E. coli and the protein thus overproduced will be purified. The interactions of DnaA protein with the DnaA boxes of the replication origin will be established in vitro by gel retardation, nitrocellulose and DNasel foot printing assays. Thus, by identifying the DNA fragments that support autonomous replication and investigating the interaction of DnaA protein with the autonomous replication sequences, this study will establish the early events in initiation of DNA replication in M. tuberculosis.
