Pohlhaus Jennifer Reineke, Long David T, O'Reilly Erin, Kreuzer Kenneth N
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Bacteriol. 2008 Aug;190(15):5239-47. doi: 10.1128/JB.00173-08. Epub 2008 Jun 6.
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.
喹诺酮类抗菌药物，如萘啶酸，作用于大肠杆菌中的DNA促旋酶。这些抑制剂结合并稳定促旋酶反应循环中一种正常情况下短暂存在的共价蛋白-DNA中间体，即切割复合物。切割复合物的稳定对于细胞杀伤是必要的，但并不充分——细胞毒性显然是由切割复合物通过一种未知机制转化为明显的DNA断裂所导致的。喹诺酮治疗以RecBC依赖的方式诱导细菌的SOS反应，这表明切割复合物以某种方式转化为双链断裂。然而，已知萘啶酸诱导SOS反应所需的唯一蛋白质是RecA和RecBC。为了确定参与对萘啶酸细胞毒性反应的其他蛋白质，我们使用dinD::lacZ报告构建体筛选了在萘啶酸处理后特异性缺乏SOS诱导的大肠杆菌突变体。从一组SOS部分组成型突变体（其47个不同基因被破坏）中，我们发现dnaQ插入突变体在对萘啶酸的SOS反应中特异性缺陷。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缺陷反应，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分析了几种其他SOS基因对萘啶酸的反应表达。一部分SOS基因在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。这些结果表明复制复合物在调节对萘啶酸的SOS反应中起作用，并且该反应比简单的开/关开关更为复杂。
