Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138.
J Biol Chem. 2013 Apr 5;288(14):10012-10023. doi: 10.1074/jbc.M112.415612. Epub 2013 Feb 12.
MutM is a bacterial DNA glycosylase that serves as the first line of defense against the highly mutagenic 8-oxoguanine (oxoG) lesion, catalyzing glycosidic bond cleavage of oxoG to initiate base excision DNA repair. Previous work has shown that MutM actively interrogates DNA for the presence of an intrahelical oxoG lesion. This interrogation process involves significant buckling and bending of the DNA to promote extrusion of oxoG from the duplex. Structural snapshots have revealed several different highly conserved residues that are prominently inserted into the duplex in the vicinity of the target oxoG before and after base extrusion has occurred. However, the roles of these helix-invading residues during the lesion recognition and base extrusion process remain unclear. In this study, we set out to probe the function of residues Phe(114) and Met(77) in oxoG recognition and repair. Here we report a detailed biochemical and structural characterization of MutM variants containing either a F114A or M77A mutation, both of which showed significant decreases in the efficiency of oxoG repair. These data reveal that Met(77) plays an important role in stabilizing the lesion-extruded conformation of the DNA. Phe(114), on the other hand, appears to destabilize the intrahelical state of the oxoG lesion, primarily by buckling the target base pair. We report the observation of a completely unexpected interaction state, in which the target base pair is ruptured but remains fully intrahelical; this structure vividly illustrates the disruptive influence of MutM on the target base pair.
MutM 是一种细菌 DNA 糖基化酶，可作为抵御高度诱变的 8-氧鸟嘌呤（oxoG）损伤的第一道防线，催化 oxoG 的糖苷键断裂，启动碱基切除 DNA 修复。先前的工作表明，MutM 主动检测 DNA 中是否存在内螺旋 oxoG 损伤。这种检测过程涉及到 DNA 的显著弯曲和扭曲，以促进 oxoG 从双链体中挤出。结构快照揭示了几个不同的高度保守的残基，这些残基在碱基挤出之前和之后，在靶 oxoG 附近明显插入双链体中。然而，这些螺旋入侵残基在损伤识别和碱基挤出过程中的作用仍不清楚。在这项研究中，我们着手研究残基 Phe(114)和 Met(77)在 oxoG 识别和修复中的作用。在这里，我们报告了包含 F114A 或 M77A 突变的 MutM 变体的详细生化和结构特征，这两种突变都显著降低了 oxoG 修复的效率。这些数据表明，Met(77)在稳定 DNA 中损伤挤出构象方面发挥着重要作用。另一方面，Phe(114)似乎通过使靶碱基对弯曲来破坏 oxoG 损伤的内螺旋状态。我们报告了一种完全出乎意料的相互作用状态的观察结果，其中靶碱基对被断裂，但仍保持完全内螺旋；这个结构生动地说明了 MutM 对靶碱基对的破坏影响。
