Protein Phosphatase 2A and Cdc7 Kinase Regulate the DNA Unwinding Element Binding Protein DUE-B in Replication Initiation [Cell Biology]
September 25th, 2014 by Gao, Y., Yao, J., Poudel, S., Romer, E., Abu-Niaaj, L., Leffak, M.
The DNA unwinding element (DUE) binding protein DUE-B binds to replication origins coordinately with the 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, while 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.