Phosphorylation-Dependent Interactions between Crb2 and Chk1 Are Essential for DNA Damage Checkpoint

In response to DNA damage, the eukaryotic genome surveillance system activates a checkpoint kinase cascade. In the fission yeast Schizosaccharomyces pombe, checkpoint protein Crb2 is essential for DNA damage-induced activation of downstream effector kinase Chk1. The mechanism by which Crb2 mediates Chk1 activation is unknown. Here, we show that Crb2 recruits Chk1 to double-strand breaks (DSBs) through a direct physical interaction. A pair of conserved SQ/TQ motifs in Crb2, which are consensus phosphorylation sites of upstream kinase Rad3, is required for Chk1 recruitment and activation. Mutating both of these motifs renders Crb2 defective in activating Chk1. Tethering Crb2 and Chk1 together can rescue the SQ/TQ mutations, suggesting that the main function of these phosphorylation sites is promoting interactions between Crb2 and Chk1. A 19-amino-acid peptide containing these SQ/TQ motifs is sufficient for Chk1 binding in vitro when one of the motifs is phosphorylated. Remarkably, the same peptide, when tethered to DSBs by fusing with either recombination protein Rad22/Rad52 or multi-functional scaffolding protein Rad4/Cut5, can rescue the checkpoint defect of crb2Δ. The Rad22 fusion can even bypass the need for Rad9-Rad1-Hus1 (9-1-1) complex in checkpoint activation. These results suggest that the main role of Crb2 and 9-1-1 in DNA damage checkpoint signaling is recruiting Chk1 to sites of DNA lesions.

当细胞遭遇DNA损伤时，真核基因组监视系统会激活检验点激酶级联反应。在粟酒裂殖酵母（Schizosaccharomyces pombe）中，检验点蛋白Crb2是DNA损伤诱导的下游效应激酶Chk1激活所必需的因子，然而Crb2介导Chk1激活的具体机制迄今尚未阐明。本研究证实，Crb2可通过直接的物理相互作用将Chk1招募至双链断裂（double-strand breaks, DSBs）位点。Crb2内存在一对保守的SQ/TQ基序，该基序为上游激酶Rad3的共有磷酸化位点，其对于Chk1的招募与激活至关重要。若同时突变这两个基序，Crb2将丧失激活Chk1的能力。将Crb2与Chk1进行锚定融合可挽救上述SQ/TQ基序突变造成的功能缺陷，提示这些磷酸化位点的核心功能是促进Crb2与Chk1之间的相互结合。体外实验表明，当其中一个SQ/TQ基序发生磷酸化时，仅包含该基序的19个氨基酸肽段即可在体外结合Chk1。值得注意的是，若将该肽段与重组蛋白Rad22/Rad52或多功能支架蛋白Rad4/Cut5融合，以将其锚定至DSBs位点，则可挽救crb2Δ的检验点功能缺陷。其中，Rad22融合蛋白甚至可绕过Rad9-Rad1-Hus1（9-1-1）复合物在检验点激活过程中的作用需求。上述结果表明，Crb2与9-1-1复合物在DNA损伤检验点信号通路中的核心作用，是将Chk1招募至DNA损伤位点。