Quinolone antibacterial drugs such as nalidixic acid target DNA gyrase in Escherichia coli. These inhibitors bind to and stabilize a normally transient covalent protein-DNA intermediate in the gyrase reaction cycle, referred to as the cleavage complex. Stabilization of the cleavage complex is necessary but not sufficient for cell killing--cytotoxicity apparently results from the conversion of cleavage complexes into overt DNA breaks by an as-yet-unknown mechanism(s). Quinolone treatment induces the bacterial SOS response in a RecBC-dependent manner, arguing that cleavage complexes are somehow converted into double-stranded breaks. However, the only proteins known to be required for SOS induction by nalidixic acid are RecA and RecBC. In hopes of identifying additional proteins involved in the cytotoxic response to nalidixic acid, we screened for E. coli mutants specifically deficient in SOS induction upon nalidixic acid treatment by using a dinD::lacZ reporter construct. From a collection of SOS partially constitutive mutants with disruptions of 47 different genes, we found that dnaQ insertion mutants are specifically deficient in the SOS response to nalidixic acid. dnaQ encodes DNA polymerase III epsilon subunit, the proofreading subunit of the replicative polymerase. The deficient response to nalidixic acid was rescued by the presence of the wild-type dnaQ gene, confirming involvement of the epsilon subunit. To further characterize the SOS deficiency of dnaQ mutants, we analyzed the expression of several additional SOS genes in response to nalidixic acid using real-time PCR. A subset of SOS genes lost their response to nalidixic acid in the dnaQ mutant strain, while two tested SOS genes (recA and recN) continued to exhibit induction. These results argue that the replication complex plays a role in modulating the SOS response to nalidixic acid and that the response is more complex than a simple on/off switch.

译文

:喹诺酮类抗菌药物,例如萘啶酸可靶向大肠杆菌中的DNA促旋酶。这些抑制剂在促旋酶反应周期中结合并稳定通常短暂的共价蛋白-DNA中间体,称为裂解复合物。裂解复合物的稳定对于杀死细胞是必要的,但还不足以使细胞毒性-细胞毒性显然是由于裂解复合物通过迄今未知的机制转化为明显的DNA断裂所致。喹诺酮治疗以RecBC依赖的方式诱导细菌SOS反应,认为裂解复合物以某种方式转化为双链断裂。但是,已知萘啶酸诱导SOS所需的唯一蛋白质是RecA和RecBC。为了希望鉴定涉及对萘啶酸的细胞毒性反应的其他蛋白质,我们使用dinD :: lacZ报告基因构建体筛选了在萘啶酸处理后特异缺乏SOS诱导作用的大肠杆菌突变体。从具有47个不同基因破坏的SOS部分组成性突变体的集合中,我们发现dnaQ插入突变体在SOS对萘啶酸的反应中特别缺乏。 dnaQ编码DNA聚合酶IIIε亚基,即复制性聚合酶的校对亚基。野生型dnaQ基因的存在挽救了对萘啶酸的不足反应,证实了ε亚基的参与。为了进一步表征dnaQ突变体的SOS缺陷,我们使用实时PCR分析了响应于萘啶酸的一些其他SOS基因的表达。 SOS基因的一个子集在dnaQ突变菌株中失去了对萘啶酸的反应,而两个测试的SOS基因(recA和recN)继续表现出诱导作用。这些结果表明复制复合物在调节SOS对萘啶酸的反应中起作用,并且该反应比简单的开/关开关更复杂。

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