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Microbiology. 2019 Oct 8. doi: 10.1099/mic.0.000859. [Epub ahead of print]

Streptomycete origin of chromosomal replication with two putative unwinding elements.

Author information

1
Department of Molecular Microbiology, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
2
Department of Microbiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.
3
Institute of Biotechnology, Faculty III, Technical University Berlin (TUB), Berlin, Germany.
4
Present address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
5
Present address: Center for Infectious Disease, Seattle, WA, USA.

Abstract

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

KEYWORDS:

DNA replication; Streptomyces; oriC

PMID:
31592764
DOI:
10.1099/mic.0.000859

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