Evidence for a chromosome origin unwinding system broadly conserved in bacteria

Abstract

Genome replication is a fundamental requirement for the proliferation of all cells. Throughout the domains of life, conserved DNA replication initiation proteins assemble at specific chromosomal loci termed replication origins and direct loading of replicative helicases (1). Despite decades of study on bacterial replication, the diversity of bacterial chromosome origin architecture has confounded the search for molecular mechanisms directing the initiation process. Recently a basal system for opening a bacterial chromosome origin (oriC) was proposed (2). In the model organism Bacillus subtilis, a pair of double-stranded DNA (dsDNA) binding sites (DnaA-boxes) guide the replication initiator DnaA onto adjacent single-stranded DNA (ssDNA) binding motifs (DnaA-trios) where the protein assembles into an oligomer that stretches DNA to promote origin unwinding. We report here that these core elements are predicted to be present in the majority of bacterial chromosome origins. Moreover, we find that the principle activities of the origin unwinding system are conserved in vitro and in vivo. The results suggest that this basal mechanism for oriC unwinding is broadly functionally conserved and therefore may represent an ancestral system to open bacterial chromosome origins.

Molecular determinants of the Bacillus subtilis chromosome origin basal unwinding system

“…Interestingly, in the crystal structure this domain IV protein:protein interaction places the helix-turn-helix motif in an orientation that would preclude dsDNA binding (Duderstadt et al, 2010;Erzberger et al, 2006) Importantly, this minimal model agrees with experimental data showing that a minimum of three DnaA-trios was necessary for B. subtilis viability (Richardson et al, 2016). Moreover, a previous bioinformatic search for BUS elements within bacterial chromosome origins set a threshold requirement of three DnaA-trios (Pelliciari et al, 2021). Therefore, the model described here would apply to all putative BUS systems identified.…”

“…BUS activity was previously reconstituted in vitro using purified DnaA proteins and fluorescently-labelled oligonucleotide scaffolds (Pelliciari et al , 2021; Richardson et al , 2019). While these experiments defined the oriC sequence elements and DnaA activities required for the BUS, they could not discriminate between models of DnaA binding dsDNA and ssDNA simultaneously (which in these reactions would occur in trans between separate DNA molecules) versus binding these elements independently (Fig.…”

“…2B). Here DnaA strand separation activity was assayed in bulk using scaffolds containing a B lack H ole Q uencher (BHQ2) and a fluorophore (Cy5); separation of the fluorescently labelled oligonucleotide from the scaffold is detected using a plate reader (Pelliciari et al , 2021).…”

“…The B. subtilis BUS does not require DnaA to bind dsDNA and ssDNA simultaneously BUS activity was previously reconstituted in vitro using purified DnaA proteins and fluorescently-labelled oligonucleotide scaffolds (Pelliciari et al, 2021;Richardson et al, 2019).…”

“…Recently, a broadly conserved chromosome origin B asal U nwinding S ystem (BUS) was reported (Fig. 1A) (Pelliciari et al , 2021). In this reaction, DnaA binding to DnaA-boxes (dsDNA binding) stimulates the assembly of DnaA ATP into an oligomer that engages DnaA-trios (ssDNA binding) to promote DNA strand separation.…”