The c-myc DNA-unwinding element-binding protein modulates the assembly of DNA replication complexes in vitro.
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 approximately 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.
Casper
John M
JM
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Kemp
Michael G
MG
Ghosh
Maloy
M
Randall
Gia M
GM
Vaillant
Andrew
A
Leffak
Michael
M
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D002845:Chromatography; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D004622:Embryo, Mammalian / Q000378:metabolism; D004625:Embryo, Nonmammalian; D004797:Enzyme-Linked Immunosorbent Assay; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D016196:S Phase; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D020798:Two-Hybrid System Techniques; D014981:Xenopus
D002843:Chromatin; D003432:Cross-Linking Reagents; D017931:DNA Primers; D004268:DNA-Binding Proteins; C500359:DTD1 protein, human; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D000604:Amino Acyl-tRNA Synthetases