No abstract

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (

Two key elements that are thought to be required for replication initiation in eubacteria are the DnaA protein, a trans-acting factor, and the replication origin, a cis-acting element. As a first step in studying the replication initiation process in mycobacteria, we have isolated a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that contains the dnaA gene. Nucleotide sequence analysis of this region revealed homologies with the rpmH gene, which codes for the ribosomal protein L34, the dnaA gene, which codes for the replication initiator protein DnaA, and the 5 end of the dnaN gene, which codes for the beta subunit of DNA polymerase III. Further, we provide evidence that when cloned into pUC18, a plasmid that is nonreplicative in M. smegmatis, the DNA fragment containing the dnaA gene and its flanking regions rendered the former capable of autonomous replication in M. smegmatis. We suggest that the M. smegmatis chromosomal origin of replication is located within the 4-kb DNA fragment.The genus Mycobacterium includes both rapid growers (e.g., Mycobacterium smegmatis and M. fortuitum) and slow growers (e.g., M. tuberculosis, M. bovis BCG, and M. leprae). The doubling time for rapid growers is approximately 3 to 4 h, compared with 20 to 24 h for slow growers. Some of the slow growers, such as M. tuberculosis and M. leprae, are major human pathogens. M. tuberculosis is a well-recognized opportunistic infectious agent for immunocompromised patients (24), and recent years have seen the emergence of M. tuberculosis strains that are resistant to one or more traditionally used antimycobacterial drugs. Our inability to control mycobacterial infections is due to a limited understanding of the basic metabolic processes, such as DNA replication and recombination, in these pathogens (33,36). An improved understanding of the key steps involved in the replication process of both rapid growers and slow growers would enable one to identify metabolic targets against which new generation drugs could be directed (2).Initiation of replication is believed to occur when DnaA, the initiator protein, interacts with the replication origin called the replicon or the replicator (15,17). Detailed genetic studies carried out in Escherichia coli (15,17) and limited studies carried out in Bacillus subtilis (9,(20)(21)(22) revealed that DnaA protein is required for replication initiation. DnaA protein initiates replication by selectively binding to repetitive units of nine-nucleotide DnaA protein recognition sequences, present in the origin of replication, called the DnaA boxes. This process triggers a cascade of events which result in replication initiation (4,15,17). In many bacteria, the dnaA gene is flanked by the rpmH and rnpA genes on the 5Ј side and the dnaN, recF, and gyrB genes on the 3Ј side (15,22,28). Exceptions to this gene order have been found in Rhizobium meliloti, Caulobacter crescentus, and Synechocystis sp. (16,25,38). Comparative analyses of the amino acid sequences of DnaA proteins from different bacteria have re...

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.The dnaA gene flanking region in many bacteria is the origin of chromosomal DNA replication, or oriC region (10,26,27). The oriC region is generally AT nucleotide rich, containing repeats of AT-rich nucleotide sequences varying in length from 13 (e.g., Escherichia coli and pseudomonads) to 16 (e.g., Bacillus subtilis) together with several 9-nucleotide DnaA protein recognition sequences called DnaA boxes (10,17,26,27). Biochemical studies with purified proteins and the oriC region of E. coli revealed that the first step in the initiation of DNA replication involved binding of 20 or more DnaA molecules to the DnaA boxes (3). This step is presumed to be followed by opening of the double-stranded oriC DNA at 13-mer AT-rich cluster sites for the entry of DnaC-directed DnaB protein.Additional events at the oriC region then result in unwinding of double-stranded DNA, priming of DNA synthesis, and formation of replication forks. Genetic studies with mutant DnaA proteins and mutant origin regions further clarified the role(s) of the DnaA protein and the mechanism of replication initiation in E. coli (10, 15).The mycobacteria exhibit varied growth rates, with the doubling times ranging from 2 to 3 h (Mycobacterium smegmatis and M. fortuitum) to 10 to 12 h (M. avium-intracellulare complex) to 22 to 24 h (M. tuberculosis and M. bovis BCG) (29). The genetic and biochemical aspects of replication, especially initiation, in mycobacteria are not well understood. As a first step toward identifying the key playe...