Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
DNA Repair (Amst). 2011 Jan 2;10(1):94-101. doi: 10.1016/j.dnarep.2010.10.005. Epub 2010 Nov 11.
Thymine deprivation results in the loss of viability in cells from bacteria to eukaryotes. Numerous studies have identified a variety of molecular processes and cellular responses associated with thymineless death (TLD). It has been observed that TLD occurs in actively growing cells, and DNA damage and DNA recombination structures have been associated with cells undergoing TLD. We measured the loss of viability in thymine-starved cells differing in the number of overlapping replication cycles (n), and we found that the magnitude of TLD correlates with the number of replication forks. By using pulsed field gel electrophoresis (PFGE), we determined the proportion of linear DNA (DSBs) and the amount of DNA remaining in the well after treatment with XbaI (nmDNA) under thymine starvation in the absence or presence of both rifampicin (suppressing TLD) and hydroxyurea (maintaining TLD). Our results indicate that DSBs and nmDNA are induced by thymine starvation, but they do not correlate with the lethality observed in the presence of the drugs. We asked whether TLD was related to chromosomal DNA initiation. DNA labeling experiments and flow cytometric analyses showed that new initiation events were induced under thymine starvation. These new DNA replication initiation events were inhibited in the presence of rifampicin but not in the presence of hydroxyurea, indicating that TLD correlates with the induction of new initiation events in Escherichia coli. In support of this finding, cells carrying a deletion of the datA site, in which DNA initiation is allowed in the presence of rifampicin, underwent TLD in the presence of rifampicin. We propose that thymineless-induced DNA initiation generates a fraction of DNA damage and/or nmDNA at origins that is critical for TLD. Our model provides new elements to be considered when testing mammalian chemotherapies that are based on the inhibition of thymidylate synthetase.
胸苷匮乏会导致从细菌到真核生物的细胞丧失活力。许多研究已经确定了与无胸腺嘧啶死亡（TLD）相关的各种分子过程和细胞反应。已经观察到 TLD 发生在生长活跃的细胞中，并且与经历 TLD 的细胞相关的是 DNA 损伤和 DNA 重组结构。我们测量了在胸腺嘧啶饥饿的细胞中丧失活力的程度，这些细胞的重叠复制周期（n）数量不同，我们发现 TLD 的幅度与复制叉的数量相关。通过使用脉冲场凝胶电泳（PFGE），我们在缺乏或存在利福平（抑制 TLD）和羟基脲（维持 TLD）的情况下，确定了 XbaI 处理后线性 DNA（DSBs）的比例和 well 中剩余 DNA 的量（nmDNA）在胸腺嘧啶饥饿下。我们的结果表明，DSBs 和 nmDNA 是由胸苷饥饿引起的，但它们与存在药物时观察到的致死性无关。我们问 TLD 是否与染色体 DNA 起始有关。DNA 标记实验和流式细胞分析表明，在胸苷饥饿下诱导了新的起始事件。这些新的 DNA 复制起始事件在利福平存在下受到抑制，但在羟基脲存在下不受抑制，表明 TLD 与大肠杆菌中诱导新的起始事件有关。支持这一发现的是，携带 datA 位点缺失的细胞，在利福平存在下允许 DNA 起始，在利福平存在下经历 TLD。我们提出，无胸腺嘧啶诱导的 DNA 起始会在起始点产生一定比例的 DNA 损伤和/或 nmDNA，这对 TLD 至关重要。我们的模型为测试基于胸苷酸合成酶抑制的哺乳动物化疗提供了新的考虑因素。
