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Molecular and Cellular Biology, May 2005, p. 3814-3830, Vol. 25, No. 9
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.9.3814-3830.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Human Stress-Activated Protein kin17 Belongs to the Multiprotein DNA Replication Complex and Associates In Vivo with Mammalian Replication Origins

Commissariat à l'Energie Atomique (CEA), Laboratoire de Génétique de la Radiosensibilité, Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, Fontenay-aux-Roses,¹ CNRS, Institut André Lwoff, Replication de l'ADN et Ultrastructure du Noyau, UPR 1983, Villejuif 94801, France,⁴ Istituto di Genetica Molecolare-CNR, Pavia, Italy,² McGill Cancer Center and Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada³

Received 23 June 2004/ Returned for modification 6 August 2004/ Accepted 21 January 2005

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G₁/S border and throughout the S phase and was negligible in both G₀ and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase . A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

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