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.高度保守的Dbf4依赖性激酶（Dbf4-Dependent Kinase, DDK）在细胞S期的细胞核中发挥关键调控作用，可直接磷酸化复制解旋酶——微染色体维持（minichromosome maintenance, MCM）复合体，进而启动染色体复制过程。然而迄今为止，除MCM复合体外，几乎未发现其他DDK的作用靶点，这使得DDK成为一类研究尚不充分的激酶。本研究报道了一种双管齐下的基于质谱（mass spectrometry）的研究策略，并鉴定得到细胞核内依赖于DDK的磷酸化蛋白质组（phosphoproteome），该蛋白质组包含约400个磷酸化事件。在该磷酸化网络中，我们发现DDK可直接磷酸化多亚基染色质重塑复合体INO80的Arp8亚基。Arp8的磷酸化能够稳定INO80的分子内复合体结构完整性，进而微调其在复制起始位点处的核小体间距调控活性。这种对复制起始位点染色质结构的调控可促进DNA复制过程，并对复制应激应答至关重要。本研究结果为我们理解复制起始位点调控的分子机制带来了重要进展。