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J. Biol. Chem., Vol. 279, Issue 52, 54529-54532, December 24, 2004

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Closing of the Fingers Domain Generates Motor Forces in the HIV Reverse Transcriptase^*

Hailong Lu, Jed Macosko, Diana Habel-Rodriguez, Rebecca W. Keller, James A. Brozik, and David J. Keller

From the Department of Chemistry, The University of New Mexico, Albuquerque, New Mexico 87106

Using the force sensor of an atomic force microscope, motor forces of the human immunodeficiency virus-1 reverse transcriptase were measured during active replication of a short DNA transcript. At low load forces the polymerase is mechanically slowed, whereas at high force (15 piconewton) it stalls. From recordings of estimated polymerase turnover velocity versus load force, an approximate force-velocity curve has been constructed. The shape of the curve suggests that load force strongly inhibits the rate-limiting step of the polymerase turnover cycle and that the combined effect of load on all steps involves an effective motion of about 1.6 nm. Earlier results from pre-steady-state kinetics experiments have identified the rate-limiting step as the closing of the fingers domain to form a tight catalytic complex. Together these findings indicate that the closing of the fingers domain is a major force-generating step for human immunodeficiency virus reverse transcriptase and, by extension, for all DNA polymerase machines.

Received for publication, June 28, 2004 , and in revised form, September 21, 2004.

^* This work was supported in part by National Institutes of Health Grant GM63808 (to D. J. K. and J. A. B.). 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.

Recipient of a Discovery Institute research fellowship.

To whom correspondence should be addressed. Tel.: 505-277-1653; Fax: 505-277-2609; E-mail: dkeller{at}unm.edu.

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