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Volume 272, Number 48, Issue of November 28, 1997 pp. 30228-30236
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The RepA Protein of Plasmid RSF1010 Is a Replicative DNA Helicase

(Received for publication, June 24, 1997, and in revised form, September 22, 1997)

Eberhard Scherzinger , Günter Ziegelin , Montserrat Bárcena , José María Carazo , Rudi Lurz and Erich Lanka

From the Max-Planck-Institut für Molekulare Genetik, Dahlem, Ihnestrasse 73, D-14195 Berlin, Germany and  Centro Nacional de Biotecnologia-CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain

The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication. RepA is one of the smallest known helicases. The protein forms a homohexamer of 29,896-Da subunits. A variety of methods were used to analyze the quaternary structure of RepA. Gel filtration and cross-linking experiments demonstrated the hexameric structure, which was confirmed by electron microscopy and image reconstruction. These results agree with recent data obtained from RepA crystals diffracting at 3.5-Å resolution (Röleke, D., Hoier, H., Bartsch, C., Umbach, P., Scherzinger, E., Lurz, R., and Saenger, W. (1997) Acta Crystallogr. Sec. D 53, 213-216). The RepA helicase has 5  3 polarity. As do most true replicative helicases, RepA prefers a tailed substrate with an unpaired 3-tail mimicking a replication fork. Optimal unwinding activity was achieved at the remarkably low pH of 5.5. In the presence of Mg²⁺ (Mn²⁺) ions, the RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP. UTP and dTTP are poor effectors. Nonhydrolyzable ATP analogues, ADP, and pyrophosphate inhibit the helicase activity, whereas inorganic phosphate does not. The presence of Escherichia coli single-stranded DNA-binding protein stimulates unwinding at physiological pH 2-3-fold, whereas the RSF1010 replicon-specific primase, RepB protein, has no effect, either in the presence or in the absence of single-stranded DNA-binding protein.

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