Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
DNA Repair (Amst). 2012 Sep 1;11(9):726-32. doi: 10.1016/j.dnarep.2012.06.005. Epub 2012 Jul 10.
Escherichia coli polymerase V (pol V/UmuD(2)'C) is a low-fidelity DNA polymerase that has recently been shown to avidly incorporate ribonucleotides (rNTPs) into undamaged DNA. The fidelity and sugar selectivity of pol V can be modified by missense mutations around the "steric gate" of UmuC. Here, we analyze the ability of three steric gate mutants of UmuC to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro, and to promote UV-induced mutagenesis and cell survival in vivo. The pol V (UmuC_F10L) mutant discriminates against rNTP and incorrect dNTP incorporation much better than wild-type pol V and although exhibiting a reduced ability to bypass a CPD in vitro, does so with high-fidelity and consequently produces minimal UV-induced mutagenesis in vivo. In contrast, pol V (UmuC_Y11A) readily misincorporates both rNTPs and dNTPs during efficient TLS of the CPD in vitro. However, cells expressing umuD'C(Y11A) were considerably more UV-sensitive and exhibited lower levels of UV-induced mutagenesis than cells expressing wild-type umuD'C or umuD'C(Y11F). We propose that the increased UV-sensitivity and reduced UV-mutability of umuD'C(Y11A) is due to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than an inability to traverse UV-induced lesions.
大肠杆菌聚合酶 V（pol V/UmuD(2)'C）是一种低保真度的 DNA 聚合酶，最近的研究表明它能够在未受损的 DNA 中强烈掺入核糖核苷酸（rNTP）。pol V 的保真度和糖选择性可以通过 UmuC 的“位阻门”周围的错义突变来修饰。在这里，我们分析了 UmuC 的三个位阻门突变体在体外促进跨损伤 DNA 合成（TLS）环丁烷嘧啶二聚体（CPD）的能力，以及在体内促进 UV 诱导的突变和细胞存活的能力。pol V（UmuC_F10L）突变体对 rNTP 和错误的 dNTP 掺入的歧视性比野生型 pol V 好得多，尽管在体外绕过 CPD 的能力降低，但它具有高保真度，因此在体内产生的 UV 诱导突变最小。相比之下，pol V（UmuC_Y11A）在体外有效地进行 CPD 的 TLS 时，很容易错误掺入 rNTP 和 dNTP。然而，表达 umuD'C(Y11A)的细胞比表达野生型 umuD'C 或 umuD'C(Y11F)的细胞对 UV 更敏感，并且 UV 诱导的突变水平更低。我们提出，umuD'C(Y11A)的 UV 敏感性增加和 UV 突变性降低是由于在 TLS 期间过度掺入 rNTP，随后这些 rNTP 被靶向修复，而不是无法穿越 UV 诱导的损伤。
