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J. Biol. Chem., Vol. 279, Issue 34, 35368-35376, August 20, 2004

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Coordinated Regulation of Replication Protein A Activities by Its Subunits p14 and p32^*

Klaus Weisshart, Pavel Pestryakov¶, Richard W. P. Smith||, Hella Hartmann, Elisabeth Kremmer**, Olga Lavrik¶, and Heinz-Peter Nasheuer

From the Institute of Molecular Biotechnology, Beutenbergstrasse 11, Jena 07745, Germany, the ^¶Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Science, Prospect Lavrentieva 8, Novosibirsk 630090, Russia, the ^**GSF, Institut für Immunologie, Marchioninistrasse 25, München 81377, Germany, and the National University of Ireland, Galway, Ireland

The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPAp14, consisting of p70 and p32. RPAp14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPAp14 complex p32 only minimally recognized the 3'-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPAp14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase -primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on single-stranded DNA requires all three subunits to support DNA replication.

Received for publication, April 6, 2004 , and in revised form, June 7, 2004.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants NA190/12-3, NA 190/13-1, SFB604, and 436-RUS-113/299/6-2, European Community Grant ERB FMRX-CT97-0125, Russian Foundation for Basic Research Grant 02-04-48404, Human Frontier Science Program RGP7/2004, Millenium Research Fund 2003 of the National University of Ireland, Galway, and Health Research Board, Ireland, Grant RP/2003/133. The Institute of Molecular Biology is a Gottfried-Wilhelm-Leibniz-Institut and is supported financially by the federal government and the Land Thüringen. 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.

Present address: Carl Zeiss Jena GmbH, Carl-Zeiss-Promenade 10, Jena 07745, Germany.

^|| Present address: University of Glasgow, Institute of Virology, Church St., Glasgow G11 5JR, Scotland, United Kingdom.

To whom correspondence should be addressed: Dept. of Biochemistry, National University of Ireland, Galway, the Cell Cycle Control Laboratory, University Rd., Galway, Ireland. Tel.: 00353-91-512-409; Fax: 00353-91-512-504; E-mail: h.nasheuer{at}nuigalway.ie.

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