MTBP phosphorylation controls DNA replication origin firing

Pedro Ferreira,Anika Marko,Verena Höfer,Dominik Boos,Nora Kronshage,Boris Pfander,Nikolai Tschernoster,Janine Altmüller,Bilal Tetik,Karl-Uwe Reusswig,Kerstin Köhler,Nina Schulze,Christoph Dießel

doi:10.1038/s41598-021-83287-w

Abstract

Faithful genome duplication requires regulation of origin firing to determine loci, timing and efficiency of replisome generation. Established kinase targets for eukaryotic origin firing regulation are the Mcm2-7 helicase, Sld3/Treslin/TICRR and Sld2/RecQL4. We report that metazoan Sld7, MTBP (Mdm2 binding protein), is targeted by at least three kinase pathways. MTBP was phosphorylated at CDK consensus sites by cell cycle cyclin-dependent kinases (CDK) and Cdk8/19-cyclin C. Phospho-mimetic MTBP CDK site mutants, but not non-phosphorylatable mutants, promoted origin firing in human cells. MTBP was also phosphorylated at DNA damage checkpoint kinase consensus sites. Phospho-mimetic mutations at these sites inhibited MTBP’s origin firing capability. Whilst expressing a non-phospho MTBP mutant was insufficient to relieve the suppression of origin firing upon DNA damage, the mutant induced a genome-wide increase of origin firing in unperturbed cells. Our work establishes MTBP as a regulation platform of metazoan origin firing.

Highlights

The eukaryotic genome must be duplicated completely and accurately to guarantee inheritance of stable genomes
In yeast, rising activities of Dbf4-dependent kinase (DDK) and cyclindependent kinase (CDK) in S phase mediate the assembly of pre-initiation complexes on pre-replicative complexes (pre-RCs), comprising Sld3-Sld[7], Sld[2], Dpb[11], DNA polymerase epsilon, Cdc[45] and GINS8,13–20
We realised that MTBP is phosphorylated at consensus sites for CDK and DNA damage checkpoint kinases

Summary

Introduction

The eukaryotic genome must be duplicated completely and accurately to guarantee inheritance of stable genomes. The main regulation step of origin firing is pre-initiation complex (pre-IC) formation as exemplified by classic cell cycle and DNA damage checkpoint regulations of replication initiation in yeast[2,12]. In yeast, rising activities of Dbf4-dependent kinase (DDK) and cyclindependent kinase (CDK) in S phase mediate the assembly of pre-initiation complexes (pre-ICs) on pre-RCs, comprising Sld3-Sld[7] (both form a constitutive complex), Sld[2], Dpb[11], DNA polymerase epsilon, Cdc[45] and GINS8,13–20. In addition to the described fundamental regulations by the cell cycle and the DNA damage checkpoint, fine tuning the levels and activities of pre-IC factors is critical for origin firing timing and replication fidelity[7,8,33,34,35]. The DNA damage kinase Mec[1] has basal activity in the absence of exogenous DNA d amage[36], and the ATR-dependent kinase pathway in metazoa was found to attenuate origin firing in the absence of induced DNA damage[37,38]

Methods

Results

Conclusion