The c-myc DNA-unwinding Element-binding Protein Modulates the Assembly of DNA Replication Complexes in Vitro

doi:10.1074/jbc.M404754200

The c-myc DNA-unwinding Element-binding Protein Modulates the Assembly of DNA Replication Complexes in Vitro^*

^‡Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435 and ^‡‡REPLICor Incorporated, Laval, Quebec H7V 4A9, Canada

§§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435. Tel.: 937-775-3125; Fax: 937-775-3730; E-mail: michael.leffak{at}wright.edu.

Abstract

The presence of DNA-unwinding elements (DUEs) at eukaryotic replicators has raised the question of whether these elements contribute to origin activity by their intrinsic helical instability, as protein-binding sites, or both. We used the human c-myc DUE as bait in a yeast one-hybrid screen and identified a DUE-binding protein, designated DUE-B, with a predicted mass of 23.4 kDa. Based on homology to yeast proteins, DUE-B was previously classified as an aminoacyl-tRNA synthetase; however, the human protein is ∼60 amino acids longer than its orthologs in yeast and worms and is primarily nuclear. In vivo, chromatin-bound DUE-B localized to the c-myc DUE region. DUE-B levels were constant during the cell cycle, although the protein was preferentially phosphorylated in cells arrested early in S phase. Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G₁ to S phase and induced cell death. DUE-B extracted from HeLa cells or expressed from baculovirus migrated as a dimer during gel filtration and co-purified with ATPase activity. In contrast to endogenous DUE-B, baculovirus-expressed DUE-B efficiently formed high molecular mass complexes in Xenopus egg and HeLa extracts. In Xenopus extracts, baculovirus-expressed DUE-B inhibited chromatin replication and replication protein A loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication. Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo.

Footnotes

↵1 The abbreviations used are: ORC, origin recognition complex; MCM, minichromosome maintenance; pre-RC, pre-replication complex; RPA, replication protein A; ARS, autonomously replicating sequence; ACS, ARS consensus sequence; DUE, DNA-unwinding element; DUE-B, DUE-binding protein; xlDUE-B, X. laevis DUE-B; siRNA, small interfering RNA; Gal4AD, Gal4 activation domain; Ni-NTA, nickel-nitrilotriacetic acid; ELISA, enzyme-linked immunosorbent assay; RSB, reticulocyte standard buffer; ChIP, chromatin immunoprecipitation; NPE, nucleoplasmic extract.
↵2 M. Ghosh, M. Ritzi, M. Kemp, A. Schepers, G. Liu, and M. Leffak, manuscript in preparation.
↵3 M. G. Kemp, unpublished data.
↵* This work was supported in part by a research challenge award from the Wright State University School of Graduate Studies and by United States Public Health Service Grant GM53819 from the NIGMS. 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.
↵§ Both authors contributed equally to this work.
↵¶ Supported by predoctoral fellowships from the Wright State University Biomedical Sciences Ph.D. program.
↵∥ Present address: Medical College of Ohio, Toledo, OH 43614.
↵** Supported by a postdoctoral fellowship from the Wright State University School of Medicine.
- Received April 28, 2004.
- Revision received January 13, 2005.
The American Society for Biochemistry and Molecular Biology, Inc.