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J. Biol. Chem., Vol. 282, Issue 47, 34229-34234, November 23, 2007

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MCM Forked Substrate Specificity Involves Dynamic Interaction with the 5'-Tail^*

Eli Rothenberg¹, Michael A. Trakselis², Stephen D. Bell, and Taekjip Ha, Investigator with the Howard Hughes Medical Institute^¶3

From the Department of Physics and the ^¶Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 and the Medical Research Council Cancer Cell Unit, Hutchison Medical Research Council Research Center, Hills Road, Cambridge CB2 2XZ, United Kingdom

The archaeal minichromosome maintenance protein MCM forms a homohexameric complex that functions as the DNA replicative helicase and serves as a model system for its eukaryotic counterpart. Here, we applied single molecule fluorescence resonance energy transfer methods to probe the substrate specificity and binding mechanism of MCM from the hyperthermophilic Archaea Sulfolobus solfataricus on various DNA substrates. S. solfataricus MCM displays a binding preference for forked substrates relative to partial or full duplex substrates. Moreover, the nature of MCM binding to Y-shaped substrates is distinct in that MCM loads on the 3'-tail while interacting with the 5'-tail likely via the MCM surface. These results provide the first elucidation of a dynamic nature of interaction between a ring-shaped helicase interacting with an opposing single-stranded DNA tail. This interaction contributes to substrate selectivity and increases the stability of the forked DNA-MCM complex, with possible implications for the MCM unwinding mechanism.

Received for publication, July 31, 2007 , and in revised form, September 10, 2007.

^* This work was supported in part by National Institutes of Health Grant GM065367 (to T. H.) and by the Medical Research Council (to S. D. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedures," Equations 1-4, Figs. 1 and 2, and a table.

¹ Supported by a European Molecular Biology Organization long-term fellowship.

² Present address: Dept. of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260.

³ To whom correspondence should be addressed: Dept. of Physics, Howard Hughes Medical Inst., University of Illinois at Urbana-Champaign, 1110 West Green St., Urbana, IL 61801. Tel.: 217-265-0717; Fax: 217-244-7187; E-mail: tjha{at}uiuc.edu.

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