title: The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity.
abstract: We report in the present study on the catalytic properties of the Deinococcus radiodurans DNA polymerase III α subunit (αDr). The αDr enzyme was overexpressed in Escherichia coli, both in soluble form and as inclusion bodies. When purified from soluble protein extracts, αDr was found to be tightly associated with E. coli RNA polymerase, from which αDr could not be dissociated. On the contrary, when refolded from inclusion bodies, αDr was devoid of E. coli RNA polymerase and was purified to homogeneity. When assayed with different DNA substrates, αDr featured slower DNA extension rates when compared with the corresponding enzyme from E. coli (E. coli DNA Pol III, αEc), unless under high ionic strength conditions or in the presence of manganese. Further assays were performed using a ssDNA and a dsDNA, whose recombination yields a DNA substrate. Surprisingly, αDr was found to be incapable of recombination-dependent DNA polymerase activity, whereas αEc was competent in this action. However, in the presence of the RecA recombinase, αDr was able to efficiently extend the DNA substrate produced by recombination. Upon comparing the rates of RecA-dependent and RecA-independent DNA polymerase activities, we detected a significant activation of αDr by the recombinase. Conversely, the activity of αEc was found maximal under non-recombination conditions. Overall, our observations indicate a sharp contrast between the catalytic actions of αDr and αEc, with αDr more performing under recombination conditions, and αEc preferring DNA substrates whose extension does not require recombination events.
authors: Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy.
authors: Lorenzo
authors: L
authors: Randi
authors: Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy.
authors: Alessandro
authors: A
authors: Perrone
authors: Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy.
authors: Mirko
authors: M
authors: Maturi
authors: Department of Medicine, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano SA, Italy.
authors: Fabrizio
authors: F
authors: Dal Piaz
authors: Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy.
authors: Michela
authors: M
authors: Camerani
authors: Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy a.hochkoeppler@unibo.it.|CSGI, University of Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy.
authors: Alejandro
authors: A
authors: Hochkoeppler
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D034301:Deinococcus / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C074904:DNA polymerase III, alpha subunit; D011938:Rec A Recombinases; D012321:DNA-Directed RNA Polymerases; D004258:DNA Polymerase III
keywords: DNA polymerase III; Deinococcus radiodurans; DnaE polymerase; Escherichia coli; RecA; α subunit