Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP

The bacteriophage phiBP contains a newly discovered putative replisome organizer, a helicase loader, and a beta clamp, which together may serve to replicate its DNA. Bioinformatics analysis of the phiBP replisome organizer sequence showed that it belongs to a recently identified family of putative initiator proteins. We prepared and isolated a wild type-like recombinant protein, gpRO-HC, and a mutant protein gpRO-HCK8A, containing a lysine to alanine substitution at position 8. gpRO-HC had low ATPase activity regardless of the presence of DNA, while the ATPase activity of the mutant was significantly higher. gpRO-HC bound to both single- and double-stranded DNA substrates. Different methods showed that gpRO-HC forms higher oligomers containing about 12 subunits. This work provides the first information about another group of phage initiator proteins, which trigger DNA replication in phages infecting low GC Gram-positive bacteria.

Automated summary

The bacteriophage phiBP contains putative replisome organizer, helicase loader, and beta clamp that may play a role in DNA replication. Bioinformatics analysis found that the phiBP replisome organizer belongs to a newly identified family of initiator proteins. Recombinant proteins gpRO-HC and gpRO-HCK8A, with a lysine to alanine substitution at position 8, were prepared and studied. gpRO-HC showed low ATPase activity, regardless of DNA presence, while the mutant demonstrated significantly higher ATPase activity. gpRO-HC bound to both single- and double-stranded DNA substrates, forming higher oligomers composed of about 12 subunits. This study provides valuable information on phage initiator proteins involved in DNA replication in low GC Gram-positive bacteria.