Streptomycete origin of chromosomal replication with two putative unwinding elements

DNA replication is controlled mostly at the initiation step. In bacteria, replication of the chromosome starts at a single origin of replication called . The initiator protein, DnaA, binds to specific sequences (DnaA boxes) within and assembles into a filament that promotes DNA double helix opening within the DNA unwinding element (DUE). This process has been thoroughly examined in model bacteria, including and but we have a relatively limited understanding of chromosomal replication initiation in other species. Here, we reveal new details of DNA replication initiation in , a group of Gram-positive soil bacteria that possesses a long linear (8-10 Mbps) and GC-rich chromosome with a centrally positioned . We used comprehensive and analyses to better characterize the structure of . We identified 14 DnaA-binding motifs and determined the consensus sequence of the DnaA box. Unexpectedly, our analysis using the WebSIDD algorithm revealed the presence of two putative DUEs (DUE1 and DUE2) located very near one another toward the 5' end of the region. P1 nuclease assay revealed that DNA unwinding occurs at both of the proposed sites, but using an replication initiation point mapping we were able to confirm only one of them (DUE2). The previously observed transcriptional activity of the region may help explain the current results. We speculate that transcription itself could modulate activity in by determining whether DNA unwinding occurs at DUE1 or DUE2.