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Papers In Press, published online ahead of print January 14, 2005
Biochemistry & Molecular Biology, Wright State University, Dayton, OH 45435
Corresponding Author: michael.leffak{at}wright.edu
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 amino acyl tRNA synthetase, however the human protein is approximately 60 amino acids longer than its orthologues in yeast or worms, and is primarily nuclear. In vivo, chromatin bound DUE-B localizes to the c myc DUE region. DUE-B levels are constant over the cell cycle although the protein is 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 migrates as a dimer during gel filtration, and co-purifies with ATPase activity. In contrast to endogenous DUE-B, baculovirus expressed DUE-B efficiently forms high molecular weight complexes in Xenopus egg or HeLa extracts. In Xenopus extracts baculovirus expressed DUE B inhibits chromatin replication and RPA 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.
J. Biol. Chem, 10.1074/jbc.M404754200
Submitted on April 28, 2004
Revised on January 13, 2005
Accepted on January 14, 2005
The c-myc DNA unwinding element binding protein modulates the assembly of DNA replication complexes in vitro
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