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 nucleosome positiong at origins of relication. Nucleosome positioning analysis was performed using MNase-seq data obtained remodeling of SGD (salt gradient dialysis) chromatin by INO80 WT and INO80-AA mutant purified protein. Futher, to test if changes in nucleosome positioning is because of DDK dependent phosphorylation, we treated our purified INO80 and INO80-AA protein with Lamda phosphatse, rephosphorylated with DDK and repurified the proteins to perform nucleosome positioing assay. We also tested nucleosome positioning in vivo in WT and ARP8 mutant strain by isolating nuclei. Atleast two biological for each experiment were performed.