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

John M Casper,Michael G Kemp,Maloy Ghosh,Gia M Randall,Andrew Vaillant,Michael Leffak

doi:10.1074/jbc.m404754200

John M Casper, Michael G Kemp + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m404754200

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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 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.

Highlights

The initiation of DNA replication in eukaryotes relies on the sequential assembly of protein complexes at replicator sequences, controlled by the activities of kinases and phospha
In S. cerevisiae, chromosomal replication origins cloned in plasmids display autonomously replicating sequence (ARS) activity and characteristically comprise a set of modular elements, including an ARS consensus sequence (ACS) [4]-binding site for origin recognition complex (ORC) [5], a region of helical instability termed a DNAunwinding element (DUE) that contributes to origin activity through template unwinding or binding of pre-replication complex (pre-RC) proteins (6 –10), and transcription factor-binding sites that can promote the assembly of replication complexes through protein-protein interactions and modification of chromatin structure
In addition to its interaction with an essential element of the c-myc replicator, DUE-B coordinately inhibited sperm chromatin replication and replication protein A (RPA) binding in the Xenopus egg extract system, and down-regulation of DUE-B slowed entry into S phase and decreased the proliferation of HeLa cells in culture

Summary

EXPERIMENTAL PROCEDURES

One-hybrid Screen—The wild-type DUE/ACS region of the c-myc origin (nucleotides 735– 832; GenBankTM/EBI accession number X00364) was cloned into vector pHisi-1 and transformed into S. cerevisiae strain YM4271 (MATa, ura, his200, ade101, lys801, leu112, trp901, tyr501, gal4-⌬512, gal80-⌬538, ade5::hisG). The DWAW reporter strain was transformed with a HeLa cDNA library cloned into pGAD-GH (Clontech) to produce Gal activation domain (Gal4AD) fusion proteins, and colonies were selected for growth at 30 °C on His/Leu-deficient medium containing 15 mM 3-aminotriazole. Plasmids were isolated from crude yeast lysates and cloned into Escherichia coli according to standard procedures. The identity of yeast strains containing mutant bait sequences was confirmed by PCR amplification and restriction enzyme digestion of the bait sequences, and these strains were transformed with a plasmid expressing the Gal4AD-DUE-B fusion protein. Doubling times in the absence of 3-aminotriazole were the same for all four reporter strains, whereas yeast transformed with a plasmid containing only Gal4AD did not grow in medium containing 2 mM 3-aminotriazole. Baculovirus-expressed recombinant DUE-B or control Sf9 cell lysates were chromatographed on Ni-NTA resin (Qiagen Inc.) under nondenaturing conditions. Binding reactions (10 mM Tris (pH 7.5), 4% glycerol, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, and 1 mM MgCl2) contained 25 fmol (2 ng) of probe, 6 pmol (0.15 ␮g) of DUE-B, and 250 ng of poly-

TABLE II ChIP PCR primers

RESULTS

DISCUSSION