Molecular insights into replication initiation in a multipartite genome harboring bacterium Deinococcus radiodurans

Deinococcus radiodurans harbors a multipartite ploid genome system consisting of two chromosomes and two plasmids present in multiple copies. How these discrete genome elements are maintained and inherited is not well understood. PprA, a pleiotropic protein involved in radioresistance, has been characterized for its roles in DNA repair, genome segregation, and cell division in this bacterium. Here, we show that PprA regulates ploidy of chromosome I and II and inhibits the activity of drDnaA, the initiator protein in D. radiodurans. We found that pprA deletion resulted in an increased genomic content and ploidy of both the chromosomal elements. Expression of PprA in trans rescued the phenotypes of the pprA mutant. To understand the molecular mechanism underlying these phenotypes, we characterized drDnaA and drDnaB. As expected for an initiator protein, recombinant drDnaA showed sequence-specific interactions with the putative oriC sequence in chromosome I (oriCI). Both drDnaA and drDnaB showed ATPase activity, also typical of initiator proteins, but only drDnaB exhibited 5′→3′ dsDNA helicase activity in vitro. drDnaA and drDnaB showed homotypic and heterotypic interactions with each other, which were perturbed by PprA. Interestingly, PprA has inhibited the ATPase activity of drDnaA but showed no effect on the activity of drDnaB. Regulation of chromosome copy number and inhibition of the initiator protein functions by PprA strongly suggest that it plays a role as a checkpoint regulator of the DNA replication initiation in D. radiodurans perhaps through its interaction with the replication initiation machinery.