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Characterization of the DdrD protein from the extremely radioresistant bacterium Deinococcus radiodurans.

ABSTRACT: Here, we report the in vitro and in vivo characterization of the DdrD protein from the extraordinary stress-resistant bacterium, D. radiodurans. DdrD is one of the most highly induced proteins following cellular irradiation or desiccation. We confirm that DdrD belongs to the Radiation Desiccation Response (RDR) regulon protein family whose expression is regulated by the IrrE/DdrO proteins after DNA damage. We show that DdrD is a DNA binding protein that binds to single-stranded DNA In vitro, but not to duplex DNA unless it has a 5' single-stranded extension. In vivo, we observed no significant effect of the absence of DdrD on the survival of D. radiodurans cells after exposure to γ-rays or UV irradiation in different genetic contexts. However, genome reassembly is affected in a ∆ddrD mutant when cells recover from irradiation in the absence of nutrients. Thus, DdrD likely contributes to genome reconstitution after irradiation, but only under starvation conditions. Lastly, we show that the absence of the DdrD protein partially restores the frequency of plasmid transformation of a ∆ddrB mutant, suggesting that DdrD could also be involved in biological processes other than the response to DNA damage.

SUBMITTER: de la Tour CB

PROVIDER: S-EPMC8254717 | 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.

Project description:Environmental DNA uptake by certain bacteria and its integration into their genome create genetic diversity and new phenotypes. DNA processing protein A (DprA) is part of a multiprotein complex and facilitates the natural transformation (NT) phenotype in most bacteria. Deinococcus radiodurans, an extremely radioresistant bacterium, is efficient in NT, and its genome encodes nearly all of the components of the natural competence complex. Here, we have characterized the DprA protein of this bacterium (DrDprA) for the known characteristics of DprA proteins of other bacteria and the mechanisms underlying the DNA-RecA interaction. DrDprA has three domains. In vitro studies showed that purified recombinant DrDprA binds to both single-strand DNA (ssDNA) and double-strand DNA (dsDNA) and is able to protect ssDNA from nucleolytic degradation. DrDprA showed a strong interaction with DrRecA and facilitated RecA-catalyzed functions in vivo. Mutational studies identified DrDprA amino acid residues crucial for oligomerization, the interaction with DrRecA, and DNA binding. Furthermore, we showed that both oligomerization and DNA binding properties of DrDprA are integral to its support of the DrRecA-catalyzed strand exchange reaction (SER) in vitro. Together, these data suggested that DrDprA is largely structurally conserved with other DprA homologs but shows some unique structure-function features like the existence of an additional C-terminal Drosophila melanogaster Miasto-like protein 1 (DML1) domain, equal affinities for ssDNA and dsDNA, and the collective roles of oligomerization and DNA binding properties in supporting DrRecA functions. IMPORTANCE Bacteria can take up extracellular DNA (eDNA) by natural transformation (NT). Many bacteria, including Deinococcus radiodurans, have constitutive competence systems and can take up eDNA throughout their growth phase. DprA (DNA processing protein A) is a transformation-specific recombination mediator protein (RMP) that plays a role in bacterial NT, and the absence of this gene significantly reduces the transformation efficiencies of both chromosomal and plasmid DNA. NT helps bacteria survive under adverse conditions and contributes to genetic diversity in bacteria. The present work describes the characterization of DprA from D. radiodurans and will add to the existing knowledge of DprA biology.

Project description:DivIVA is a member of the Min family of proteins that spatially regulates septum formation at the midcell position and cell pole determination in Bacillus subtilis. Deinococcus radiodurans, a Gram-positive coccus-shaped bacterium, is characterized by its extreme resistance to DNA-damaging agents, including radiation. D. radiodurans cells exposed to gamma radiation undergo cell division arrest by as-yet-uncharacterized mechanisms. divIVA is shown to be an essential cell division gene in this bacterium, and DivIVA of D. radiodurans (drDivIVA) interacts with genome segregation proteins through its N-terminal region. Earlier, RqkA, a gamma radiation-responsive Ser/Thr quinoprotein kinase, was characterized for its role in radioresistance in D. radiodurans. Here, we showed that RqkA phosphorylates drDivIVA at the threonine 19 (T19) residue. The phospho-mimetic mutant with a mutation of T19 to E19 in DivIVA (DivIVAT19E) is found to be functionally different from the phospho-ablative mutant (DivIVAT19A) or the wild-type drDivIVA. A DivIVAT19E-red fluorescent protein (RFP) fusion expressed in the wild-type background showed the arrest in the typical dynamics of drDivIVA and the loss of its interaction with the genome segregation protein ParA2. The allelic replacement of divIVA with divIVAT19E-rfp was not tolerated unless drDivIVA was expressed episomally, while there was no phenotypic change when the wild-type allele was replaced with either divIVAT19A-rfp or divIVA-rfp. These results suggested that the phosphorylation of T19 in drDivIVA by RqkA affected its in vivo functions, which may contribute to the cell cycle arrest in this bacterium. IMPORTANCE Deinococcus radiodurans, a radioresistant bacterium, lacks LexA/RecA-mediated DNA damage response and cell cycle regulation as known in other bacteria. However, it adjusts its transcriptome and proteome upon DNA damage. In eukaryotes, the DNA damage response and cell cycle are regulated by Ser/Thr protein kinases. In D. radiodurans, we characterized a gamma radiation-responsive Ser/Thr quinoprotein kinase (RqkA) that phosphorylated DNA repair and cell division proteins in this bacterium. In previous work, the effect of S/T phosphorylation by RqkA on activity improvement of the DNA repair proteins has been demonstrated. This study reports that Ser phosphorylation by RqkA attenuates the function of a cell polarity and plane of cell division-determining protein, DivIVA, and its cellular dynamics in response to DNA damage, which might help to understand the mechanism of cell cycle regulation in this bacterium.

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Characterization of the DdrD protein from the extremely radioresistant bacterium Deinococcus radiodurans.

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