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.

染色质组装因子I（Chromatin Assembly Factor I, CAF-I）在核小体的复制偶联组装过程中发挥关键作用。已有研究推测其功能与细胞周期调控相关，但相关细节仍较为匮乏。现有模型表明，CAF-I可通过其Cac1p亚基与复制滑动夹增殖细胞核抗原（PCNA）的相互作用，被招募至复制叉与染色质中，且该相互作用可受到激酶CDC7的促进。本研究证实，另一激酶CDC28可在S期早期将Cac1p的丝氨酸94与丝氨酸515位点磷酸化，并调控其与染色质的结合，但不影响其与PCNA的结合。针对CDC28介导的Cac1p磷酸化位点（而非CDC7的靶位点）的突变，可显著降低Cac1p的体内磷酸化水平。然而，Cac1p上推定的CDC7靶位点突变则会降低其蛋白稳定性。在cdc28–1突变体中，CAF-I与染色质的结合能力受损；而在cdc7–1突变体中，该结合能力的受损程度相对较轻。此外，同时针对CDC28与CDC7介导的Cac1p磷酸化位点进行突变，会削弱端粒区域的基因沉默效应。我们提出，这种磷酸化修饰代表了S期早期CAF-I被招募至染色质过程中的一个调控步骤，该步骤独立于CAF-I与PCNA的结合。因此，我们证实CDC28参与了DNA复制过程中染色质重组装的调控。本研究发现为细胞周期调控、DNA复制与染色质重组装之间的关联提供了全新的机制层面见解。