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

ABSTRACT: 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.

SUBMITTER: Maurya GK

PROVIDER: S-EPMC7988490 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEnzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of gamma-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair.ResultsIn this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II) and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation.ConclusionDeinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II) as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a member of the nucleotidyltransferase family. Characterization of another protein from the same operon revealed a 5'-polynucleotide kinase with a possible role in DNA strand-break repair.

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

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