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J. Biol. Chem., Vol. 280, Issue 51, 41852-41863, December 23, 2005

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Biochemical and Mutational Analyses of a Unique Clamp Loader Complex in the Archaeon Methanosarcina acetivorans^*

Yi-Hsing Chen, Svetlana A. Kocherginskaya, Yuyen Lin, Binjon Sriratana, Angelica M. Lagunas, Justin B. Robbins1, Roderick I. Mackie, and Isaac K. O. Cann2

From the Department of Animal Sciences and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Clamp loaders orchestrate the switch from distributive to processive DNA synthesis. Their importance in cellular processes is underscored by their conservation across all forms of life. Here, we describe a new form of clamp loader from the archaeon Methanosarcina acetivorans. Unlike previously described archaeal clamp loaders, which are composed of one small subunit and one large subunit, the M. acetivorans clamp loader comprises two similar small subunits (M. acetivorans replication factor C small subunit (MacRFCS)) and one large subunit (MacRFCL). The relatedness of the archaeal and eukaryotic clamp loaders (which are made up of four similar small subunits and one large subunit) suggests that the M. acetivorans clamp loader may be an intermediate form in the archaeal/eukaryotic sister lineages. The clamp loader complex reconstituted from the three subunits MacRFCS1, MacRFCS2, and MacRFCL stimulated DNA synthesis by a cognate DNA polymerase in the presence of its sliding clamp. We used site-directed mutagenesis in the Walker A and SRC motifs to examine the contribution of each subunit to the function of the M. acetivorans clamp loader. Although mutations in MacRFCL and MacRFCS2 did not impair clamp loading activity, any mutant clamp loader harboring a mutation in MacRFCS1 was devoid of the clamp loading property. Mac-RFCS1 is therefore critical to the clamp loading activity of the M. acetivorans clamp loader. It is our anticipation that the discovery of this unique replication factor C homolog will lead to critical insights into the evolution of more complex clamp loaders from simpler ones as more complex organisms evolved in the archaeal/eukaryotic sister lineages.

Received for publication, August 8, 2005 , and in revised form, October 13, 2005.

^* This work was supported in part by National Science Foundation Grant MCB-0238451 (to I. K. O. C.). 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. 1–3.

¹ Supported by Agricultural Genome Sciences and Public Policy Training Grant 2001-52100-11527.

² To whom correspondence should be addressed: Dept. of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Dr., Urbana, IL 61801. Tel.: 217-333-2090; Fax: 217-333-8286; E-mail: icann{at}uiuc.edu.