Abstract
DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004’s DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes.
Highlights
The ability to maintain, replicate, and express genetic information encoded in DNA is critical to all domains of life
Most of the initial studies on bacterial DNA replication were performed in Escherichia coli and Bacillus subtilis
Rv0004 is essential for DNA replication in mycobacteria course from Tet-DciA cultures grown -ATc, with each point representing n = 4 except the 16 h and 27 h points, which each represent n = 3. (E) dciAMtb transcript levels at 16 h growth -ATc compared to 3 h
Summary
The ability to maintain, replicate, and express genetic information encoded in DNA is critical to all domains of life. Initiation begins when DnaA, the initiator protein, binds to specific sites located at the origin of replication (oriC) and oligomerizes, forming a nucleoprotein complex that results in the melting of the adjacent DNA [1]. Helicase loaders and accessory primosomal proteins, with the help of DnaA, load the replicative helicase onto melted DNA [2,3]. The replicative helicase binds the primase, which lays down short RNA primers [2]. Clamp loader complexes load DNA Polymerase III (Pol III) onto primed DNA, allowing replication elongation to begin [1]. Elongation proceeds bi-directionally from oriC until it reaches termination sites bound by terminator proteins [4]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
