Delineation of the role of chromatin assembly and the Rtt101Mms1 E3 ubiquitin ligase in DNA damage checkpoint recovery in budding yeast

The DNA damage checkpoint is activated in response to DNA double-strand breaks (DSBs). We had previously shown that chromatin assembly mediated by the histone chaperone Asf1 triggers inactivation of the DNA damage checkpoint in yeast after DSB repair, also called checkpoint recovery. Here we show that chromatin assembly factor 1 (CAF-1) also contributes to chromatin reassembly after DSB repair, explaining its role in checkpoint recovery. Towards understanding how chromatin assembly promotes checkpoint recovery, we find persistent presence of the damage sensors Ddc1 and Ddc2 after DSB repair in asf1 mutants. The genes encoding the E3 ubiquitin ligase complex Rtt101Mms1 are epistatic to ASF1 for survival following induction of a DSB, and Rtt101Mms1 are required for checkpoint recovery after DSB repair but not for chromatin assembly. By contrast, the Mms22 substrate adaptor that is degraded by Rtt101Mms1 is required for DSB repair per se. Deletion of MMS22 blocks loading of Rad51 at the DSB, while deletion of ASF1 or RTT101 leads to persistent Rad51 loading. We propose that checkpoint recovery is promoted by Rtt101Mms1-mediated ubiquitylation of Mms22 in order to halt Mms22-dependent loading of Rad51 onto double-stranded DNA after DSB repair, in concert with the chromatin assembly-mediated displacement of Rad51 and checkpoint sensors from the site of repair.

DNA损伤检验点（DNA damage checkpoint）会响应DNA双链断裂（DNA double-strand breaks, DSBs）而被激活。本课题组前期已证实，由组蛋白伴侣Asf1（histone chaperone Asf1）介导的染色质组装，可触发酵母中DNA双链断裂修复后DNA损伤检验点的失活，该过程亦被称为检验点恢复。本研究证实，染色质组装因子1（chromatin assembly factor 1, CAF-1）同样参与DNA双链断裂修复后的染色质重组装过程，这解释了其在检验点恢复中的作用机制。为阐明染色质组装促进检验点恢复的具体机制，我们发现在asf1突变体中，DNA双链断裂修复完成后，损伤传感器Ddc1与Ddc2仍持续滞留于修复位点。编码E3泛素连接酶复合物Rtt101Mms1（E3 ubiquitin ligase complex Rtt101Mms1）的基因在诱导DNA双链断裂后的细胞存活方面与ASF1存在上位性关系，且Rtt101Mms1是DNA双链断裂修复后检验点恢复所必需的，但并非染色质组装过程所必需。与之相对，被Rtt101Mms1降解的Mms22底物适配子，本身是DNA双链断裂修复所必需的。删除MMS22会阻断Rad51在DNA双链断裂位点的加载，而删除ASF1或RTT101则会导致Rad51的持续加载。我们提出如下假说：Rtt101Mms1介导的Mms22泛素化可促进检验点恢复，从而在DNA双链断裂修复完成后，终止Mms22依赖的Rad51向双链DNA的加载；该过程与染色质组装介导的Rad51及检验点传感器从修复位点的解离协同发挥作用。