Interaction between DUE-B and Treslin is required to load Cdc45 on chromatin in human cells.

A key step in the initiation of eukaryotic DNA replication is the binding of the activator protein Cdc45 to promote MCM helicase unwinding of the origin template. We show here that the c-myc origin DNA unwinding element-binding protein, DUE-B, interacts in HeLa cells with the replication initiation protein Treslin to allow Cdc45 loading onto chromatin. The chromatin loading of DUE-B and Treslin are mutually dependent, and the DUE-B-Treslin interaction is cell cycle-regulated to peak as cells exit G1 phase prior to the initiation of replication. The conserved C-terminal domain of DUE-B is required for its binding to TopBP1, Treslin, Cdc45, and the MCM2-7 complex, as well as for the efficient loading of Treslin, Cdc45, and TopBP1 on chromatin. These results suggest that DUE-B acts to identify origins by MCM binding and serves as a node for replication protein recruitment and Cdc45 transfer to the prereplication complex.

Protein phosphatase 2A and Cdc7 kinase regulate the DNA unwinding element-binding protein in replication initiation.

The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2-7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2-7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.