Dbf4-Dependent Kinase Finetunes INO80 Function at Chromosome Replication Origins

The highly conserved Dbf4-Dependent Kinase (DDK) plays a pivotal role in the nucleus during S phase, where it directly phosphorylates the replicative helicase, the minichromosome maintenance (MCM) complex. This leads to the initiation of chromosome replication. However, aside from the MCM complex, few other targets have been identified to date, leaving DDK an understudied kinase. Here, we describe a two-pronged mass spectrometry-based approach and define the nuclear DDK-dependent phosphoproteome, which consists of approximately 400 phosphorylation events. Within this network, we found that DDK directly phosphorylates the Arp8 subunit of the multi-subunit chromatin remodeler complex INO80. Arp8 phosphorylation stabilises INO80's intramolecular complex integrity, which finetunes its nucleosome spacing activity at replication origins. This adjustment of origin chromatin architecture stimulates replication and is important for the response to replication stress. Our results represent a significant advance in our understanding of the molecular mechanisms underlying the regulation of replication origins. Overall design: It was tested whether DDK phosphorylation of INO80 might influence the transcriptome genome-wide. Gene expression profiling analysis was performed using RNAseq data obtained from isogenic ARP8 wild-type and arp-8-P (serine 65 to alanine, serine 233 to alanine) strains. Three biological replicates were performed. Another experiment analysed transcription in synchronised cells. Cells were arrested with alpha factor and harvested in G1 and early S phase.