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

Abstract

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 α. 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.

ACKNOWLEDGMENTS

This article is dedicated to the memory of Gerald B. Price (McGill Cancer Center, McGill University, Montreal, Canada).

We particularly thank Y. Frobert, C. Créminon, J. Grassi, and all the staff of the Service de Pharmacologie et d'Immunologie (DRM, CEA) for monoclonal antibodies against human kin17 protein; G. Baldacci and I. Tratner (Institut Curie, Orsay) for stimulating discussions, advice, and purified polyclonal antibody against PCNA; and E. Pichard for electron microscopy. B. Dutrillaux is gratefully acknowledged for his continuous support. We are grateful to S. Linn (Berkeley, CA) and A. M. Holmes (Waltham, MA) for providing DNA polymerase α antibody and B. Stillman for RFCp140 antibody.

Maria Zannis-Hadjopoulos, Olivia Novac, and Domenic Di Paola were supported by grants from the CIHR and FRSQ, respectively. This work was supported by Electricité de France (EDF) contract 8702.