CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase <i>CDC7</i>. Here we show that another kinase, <i>CDC28</i>, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of <i>CDC28</i> but not of <i>CDC7</i> substantially reduce the <i>in vivo</i> phosphorylation of Cac1p. However, mutations in the putative <i>CDC7</i> target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a <i>cdc28–1</i> mutant and to a lesser extent in a <i>cdc7–1</i> mutant. In addition, mutations in the Cac1p-phosphorylation sites by both <i>CDC28</i> and <i>CDC7</i> reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate <i>CDC28</i> in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.