Department of Environmental Genomics, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Mutagenesis. 2011 Mar;26(2):303-8. doi: 10.1093/mutage/geq093. Epub 2010 Nov 8.
Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70-75°C. It does not have a gene encoding O(6)-alkylguanine-DNA alkyltransferase (AGT) for the repair of O(6)-methylguanine (O(6)-meG), but it has a homologous gene atl encoding alkyltransferase-like (ATL) proteins in which the cysteine residue in the active site of the PCHR motif conserved in AGT is replaced by alanine (i.e. lack of methyltransferase activity). To investigate the role of ATL protein in the repair of O(6)-meG, we isolated atl deletion mutants and measured specific G:C→A:T transition mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by a His(+) reversion system at the hisD3110 locus. MNNG caused an increased mutation frequency in the atl-deficient mutant but a significantly higher frequency increase in a uvrA mutant, which is deficient in nucleotide excision repair (NER), indicating that both ATL protein and NER played an important role in preventing G:C→A:T transitions. We observed no difference in MNNG sensitivity between the uvrA atl double mutant and the parent uvrA strain. Our results support a recently proposed repair model in which ATL protein acts as a sensor of O(6)-meG damage and recruits UvrA protein to repair the lesion via an NER system. In addition, the finding that the uvrA atl strain mutated with greater frequency than the single atl strain suggests that O(6)-meG is repaired by NER in the absence of ATL protein. We also discuss the possible association of a transcription-repair coupling factor in a transcription-coupled repair pathway and of MutS protein in a mismatch repair pathway with ATL/NER-mediated repair of O(6)-meG.
嗜热栖热菌是一种极端嗜热的真细菌，最适生长温度为 70-75°C。它没有编码 O(6)-烷基鸟嘌呤-DNA 烷基转移酶 (AGT) 的基因，用于修复 O(6)-甲基鸟嘌呤 (O(6)-meG)，但它有一个同源基因 atl，编码具有烷基转移酶样 (ATL) 蛋白的基因，其中 PCHR 基序中活性位点的半胱氨酸残基被丙氨酸取代（即缺乏甲基转移酶活性）。为了研究 ATL 蛋白在修复 O(6)-meG 中的作用，我们分离了 atl 缺失突变体，并通过 His(+)回复系统在 hisD3110 基因座上测量了 N-甲基-N'-硝基-N-亚硝基胍 (MNNG) 诱导的特定 G:C→A:T 转换突变。MNNG 在 atl 缺陷突变体中引起突变频率增加，但在核苷酸切除修复 (NER) 缺陷的 uvrA 突变体中引起的突变频率增加更为显著，表明 ATL 蛋白和 NER 在防止 G:C→A:T 转换中都发挥了重要作用。我们在 uvrA atl 双突变体和亲本 uvrA 菌株之间观察到 MNNG 敏感性没有差异。我们的结果支持了最近提出的修复模型，即 ATL 蛋白作为 O(6)-meG 损伤的传感器，通过 NER 系统招募 UvrA 蛋白修复损伤。此外，发现 uvrA atl 菌株比单一 atl 菌株突变频率更高，这表明在没有 ATL 蛋白的情况下，NER 修复 O(6)-meG。我们还讨论了转录偶联修复途径中的转录修复偶联因子和错配修复途径中的 MutS 蛋白与 ATL/NER 介导的 O(6)-meG 修复之间的可能关联。
