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J. Biol. Chem., Vol. 282, Issue 37, 27076-27085, September 14, 2007

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Bacillus stearothermophilus PcrA Monomer Is a Single-stranded DNA Translocase but Not a Processive Helicase in Vitro^*

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110-1093

Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3' to 5' direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.

Received for publication, May 29, 2007 , and in revised form, July 11, 2007.

^* This work was supported in part by National Institutes of Health Grant GM45948. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Supported in part by Molecular Biophysics Training Grant T32 GM08492.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, WA University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. Tel.: 314-362-4393; Fax: 314-362-7183; E-mail: lohman{at}biochem.wustl.edu.

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