Search

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

SMC complexes, loaded at ParB- parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis , ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.

Replication and segregation of the genetic information is necessary for a cell to proliferate. In Bacillus subtilis , the Par system (ParA/Soj, ParB/Spo0J and parS ) is required for segregation of the chromosome origin ( oriC ) region and for proper control of DNA replication initiation. ParB binds parS sites clustered near the origin of replication and assembles into sliding clamps that interact with ParA to drive origin segregation through a diffusion-ratchet mechanism. As part of this dynamic process, ParB stimulates ParA ATPase activity to trigger its switch from an ATP-bound dimer to an ADP-bound monomer. In addition to its conserved role in DNA segregation, ParA is also a regulator of the master DNA replication initiation protein DnaA. We hypothesized that in B. subtilis the location of the Par system proximal to oriC would be necessary for ParA to properly regulate DnaA. To test this model, we constructed a range of genetically modified strains with altered numbers and locations of parS sites, many of which perturbed chromosome origin segregation as expected. Contrary to our hypothesis, the results show that regulation of DNA replication initiation by ParA is maintained when a parS site is separated from oriC . Because a single parS site is sufficient for proper control of ParA, the results are consistent with a model where ParA is efficiently regulated by ParB sliding clamps following loading at parS .

The prokaryotic nucleoid-associated protein (NAP) HU is both highly conserved and ubiquitous. Deletion of HU causes pleiotropic phenotypes, making it difficult to uncover the critical functions of HU within a bacterial cell. In their recent work, Karaboja and Wang (J Bacteriol 204:e00119-22, 2022, https://doi.org/10.1128/JB.00119-22) show that one essential function of Bacillus subtilis HU (HBsu) is to drive the DnaA-dependent initiation of DNA replication at the chromosome origin. We discuss the possible roles of HBsu in replication initiation and other essential cellular functions.

Research over the last two decades has revealed that bacterial genomes are highly organized and that bacteria have sophisticated mechanisms in place to ensure their correct replication and segregation into progeny cells. Here we discuss techniques that can be used with live bacterial cells to analyze DNA replisome dynamics, double-strand chromosome breaks, and restart of repaired replication forks., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle., (© 2021. The Author(s).)