Role of RecA and the SOS Response in Thymineless Death in <em>Escherichia coli</em>

Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans.胸腺嘧啶缺失致死（Thymineless death, TLD）是一种经典且仍未被完全阐明的神秘现象，已在细菌、酵母及人类细胞中被观测记录，指细胞在缺乏胸腺嘧啶时会快速丧失存活能力。尽管该现象是多种重要化疗药物的核心作用机制，且历经数十年的广泛研究，其核心分子机制至今仍未被阐明。在大肠杆菌（Escherichia coli）中，同源重组（homologous recombination, HR）通路的多种蛋白是胸腺嘧啶缺失致死过程所必需的；但令人意外的是，作为同源重组核心蛋白与SOS DNA损伤应答（SOS DNA–damage response）激活因子的RecA，此前被报道并非该过程的必需因子。本研究证实，RecA与SOS应答是胸腺嘧啶缺失致死过程中相当一部分细胞死亡事件的必要条件。我们发现，此前被认为与该过程相关的部分Rec家族蛋白，通过促进SOS应答的激活来推动胸腺嘧啶缺失致死进程；此外，在约40个受SOS通路上调的蛋白中，作为SOS诱导型细胞分裂抑制剂的SulA，主导了SOS通路介导的胸腺嘧啶缺失致死的绝大部分乃至全部过程。研究数据表明，胸腺嘧啶缺失致死的大部分效应源于细胞分裂受阻后引发的不可逆细胞周期检查点激活。对发生胸腺嘧啶缺失致死的细胞进行荧光原位杂交（fluorescence in situ hybridization, FISH）分析显示，细胞出现复制阻滞与明显的DNA丢失：复制起点附近区域最初即出现丰度降低，而复制终点附近区域则在后续发生丢失。本文还讨论了涉及受阻复制叉处单链DNA形成、SulA介导的细胞周期阻滞、RecQ/RecJ催化的DNA降解以及同源重组的多种作用模型。本研究数据提示，DNA损伤应答与同源重组网络在人类胸腺嘧啶缺失致死以及化疗耐药性中具有关键意义。