Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.
PLoS One. 2010 May 27;5(5):e10862. doi: 10.1371/journal.pone.0010862.
Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.
大肠杆菌有五种 DNA 聚合酶，其中低保真度的 Pol IV 或 DinB，是在研究充分的 Lac 移框回复试验中应激诱导突变所必需的。尽管 Pol IV 通常在每个细胞中存在约 200 个分子，但只有当至少两种细胞应激反应被激活时，它才会被招募到 DNA 双链断裂修复中，并引起突变：SOS DNA 损伤反应，该反应将 DinB 上调约 10 倍，以及 RpoS 控制的一般应激反应，该反应将 Pol IV 上调约 2 倍。DNA Pol III 也被牵连，但它在突变中的作用尚不清楚。我们在体内寻找应激诱导突变过程中多种 DNA 聚合酶存在和相互作用的证据。使用多重突变株，我们提供了 DNA Pols I、II 和 III 与 Pol IV 竞争的证据，这意味着它们都存在于应激诱导突变的部位。先前的数据表明 Pol V 也存在。我们表明，Pol I、II 和 III 与 Pol IV 的相互作用既不是由于当特定的 DNA 聚合酶被去除时 SOS 反应的诱导，也不是由于 Pol I 或 Pol III 的编辑功能对 Pol IV 错误的校对。第三，我们提供的证据表明，Pol III 本身不会协助，而是抑制 Pol IV 依赖性突变。这些数据支持了另一个假设，即在 DNA 双链断裂 (DSB) 修复过程中，以前在 Lac 系统中显示应激诱导突变的基础，DNA Pols I、II 和 III 与 DNA Pol IV 竞争在引物末端起作用。Pol IV 的上调，以及可能的其他应激反应控制因子，使竞争有利于易错的 Pol IV，而牺牲更准确的聚合酶，从而产生应激诱导的突变。这种突变检测试验揭示了应激时 DSB 修复中起作用的 DNA 聚合酶，也为体内 DNA 聚合酶竞争和选择提供了一个敏感的指标。
