Metabolic remodeling maintains a reducing environment for rapid activation of replication checkpoint

Nucleotide metabolism fuels normal DNA replication and is also primarily targeted by the DNA replication checkpoint when replication stalls. To reveal a comprehensive interconnection between genome maintenance and metabolism, we analyze the metabolomic changes upon replication stress in the budding yeast S. cerevisiae. We found that upon treatment of cells with hydroxyurea, glucose is rapidly diverted to the oxidative pentose phosphate pathway (PPP). This effect is mediated by the AMP-dependent kinase, SNF1, which phosphorylates the transcription factor Mig1, thereby relieving repression of the gene encoding the rate-limiting enzyme of the PPP. Surprisingly, NADPH produced by the PPP is required for efficient recruitment of replication protein A (RPA) to single-stranded DNA, providing the signal for the activation of the Mec1/ATR-Rad53/CHK1 checkpoint signaling kinase cascade. Thus SNF1, best known as a central energy controller, determines a fast mode of replication checkpoint activation through a redox mechanism. These findings establish that SNF1 provides a hub with direct links to cellular metabolism, redox and surveillance of DNA replication in eukaryotes.

核苷酸代谢可为正常DNA复制提供能量支撑，且在复制停滞时，亦是DNA复制检查点的主要作用靶点。为全面解析基因组维持与细胞代谢间的广泛关联，本研究针对酿酒酵母（Saccharomyces cerevisiae，简称S. cerevisiae）在复制应激状态下的代谢组变化展开分析。研究发现，当细胞经羟基脲处理后，葡萄糖会快速转向氧化磷酸戊糖途径（oxidative pentose phosphate pathway, PPP）；该效应由AMP依赖的蛋白激酶（AMP-dependent kinase）SNF1介导：该激酶可磷酸化转录因子Mig1，从而解除对PPP途径限速酶编码基因的转录抑制。尤为意外的是，PPP途径生成的还原型烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）是复制蛋白A（replication protein A, RPA）高效招募至单链DNA的必要条件，该过程可为Mec1/ATR-Rad53/CHK1检查点激酶级联信号通路的激活提供触发信号。由此可见，作为经典核心能量调控因子的SNF1，可通过氧化还原机制调控复制检查点的快速激活模式。本研究结果证实，SNF1在真核生物中可作为核心枢纽，直接关联细胞代谢、氧化还原稳态与DNA复制监视过程。