A Novel Checkpoint and RPA Inhibitory Pathway Regulated by Rif1

Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA–damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism. However, ssDNA has to accumulate above a certain threshold to activate checkpoints. What determines this checkpoint-activation threshold is an important, yet unanswered question. Here we identify Rif1 (Rap1-Interacting Factor 1) as a threshold-setter. Following telomere uncapping, we show that budding yeast Rif1 has unprecedented effects for a protein, inhibiting the recruitment of checkpoint proteins and RPA (Replication Protein A) to damaged chromosome regions, without significantly affecting the accumulation of ssDNA at those regions. Using chromatin immuno-precipitation, we provide evidence that Rif1 acts as a molecular “band-aid” for ssDNA lesions, associating with DNA damage independently of Rap1. In consequence, small or incipient lesions are protected from RPA and checkpoint proteins. When longer stretches of ssDNA are generated, they extend beyond the junction-proximal Rif1-protected regions. In consequence, the damage is detected and checkpoint signals are fired, resulting in cell cycle arrest. However, increased Rif1 expression raises the checkpoint-activation threshold to the point it simulates a checkpoint knockout and can also terminate a checkpoint arrest, despite persistent telomere deficiency. Our work has important implications for understanding the checkpoint and RPA–dependent DNA–damage responses in eukaryotic cells.当端粒封盖、DNA复制或DNA修复过程受阻时，细胞会积累单链DNA（single-stranded DNA，ssDNA）。这类单链DNA的积累会通过激活参与癌症防护的DNA损伤检验点，引发细胞周期阻滞。因此，单链DNA积累可能是一种抗癌机制。然而，单链DNA的积累必须达到特定阈值才能激活检验点。究竟是什么决定了这一检验点激活阈值，是一个尚未解决的重要科学问题。本研究鉴定出Rif1（Rap1-Interacting Factor 1）为阈值调控因子。在端粒脱封盖后，我们发现酿酒酵母中的Rif1展现出前所未有的蛋白功能：它能够抑制检验点蛋白与复制蛋白A（Replication Protein A，RPA）向受损染色体区域的募集，却不会显著影响这些区域内单链DNA的积累。通过染色质免疫沉淀（chromatin immuno-precipitation）技术，我们提供证据表明，Rif1可作为单链DNA损伤的分子“创可贴”，独立于Rap1与DNA损伤位点结合。因此，小型或初期损伤会被遮蔽，无法被复制蛋白A与检验点蛋白识别。当产生更长片段的单链DNA时，这些单链DNA会延伸至Rif1保护的连接点邻近区域之外。此时损伤会被检测到，检验点信号得以激活，进而引发细胞周期阻滞。然而，Rif1表达上调会提升检验点激活阈值，直至其模拟出检验点敲除的效果；即便端粒缺陷持续存在，该上调也可终止检验点介导的细胞周期阻滞。本研究对于理解真核细胞中依赖于检验点与复制蛋白A的DNA损伤应答机制具有重要意义。