Abstract
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.
Highlights
Cdc7 kinase and protein phosphatase 2A are important in DNA replication initiation
DNA unwinding element (DUE)-B Is Incorporated into High Molecular Weight (HMW) Complexes during M Phase—To test the hypothesis that phosphorylation of DUE-B could change its ability to interact with additional proteins, the formation of complexes containing DUE-B was assayed by size exclusion chromatography of cell extracts
DUE-B is required for Cdc45 binding to chromatin in Xenopus egg extract, and the loading of endogenous DUE-B and Cdc45 depends on pre-replication complex (pre-RC) assembly and S phase kinase activity
Summary
Cdc kinase and protein phosphatase 2A are important in DNA replication initiation. Results: DNA unwinding element (DUE)-binding protein (DUE-B) is an essential replication factor that is regulated by Cdc and PP2A. The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc in vivo, and loads Cdc onto chromatin in Xenopus egg extracts. Unwinding of eukaryotic DNA for replication initiation requires the sequential binding of multiple proteins onto replication origins, resulting in activation of the minichromosome maintenance (MCM) replicative helicase This process is separated between the M/G1 and S phases of the cell cycle such that the origin recognition complex recognizes replication origins in late M/G1 phase to serve as a platform for the recruitment of additional replication proteins [1]. The requirement of DDK for cell survival can be bypassed by mutation of the MCM subunits, suggesting that the Mcm complex is an essential target of DDK [9, 10] These bypass mutants show growth defects in the absence of DDK activity, implying that there may be other factors phosphorylated by DDK for efficient DNA replication. The same mutant increases MCM binding but inhibits Cdc45/RPA loading onto chromatin and cell division in HeLa cells
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