Mrr from Escherichia coli K12 is a type IV restriction endonuclease whose role is to recognize and cleave foreign methylated DNA. Beyond this protective role, Mrr can inflict chromosomal DNA damage that elicits the SOS response in the host cell upon heterologous expression of specific methyltransferases such as M.HhaII, or after exposure to high pressure (HP). Activation of Mrr in response to these perturbations involves an oligomeric switch that dissociates inactive homo-tetramers into active dimers. Here we used scanning number and brightness (sN&B) analysis to determine in vivo the stoichiometry of a constitutively active Mrr mutant predicted to be dimeric and examine other GFP-Mrr mutants compromised in their response to either M.HhaII activity or HP shock. We also observed in vitro the direct pressure-induced tetramer dissociation by HP fluorescence correlation spectroscopy of purified GFP-Mrr. To shed light on the linkages between subunit interactions and activity of Mrr and its variants, we built a structural model of the full-length tetramer bound to DNA. Similar to functionally related endonucleases, the conserved DNA cleavage domain would be sequestered by the DNA recognition domain in the Mrr inactive tetramer, dissociating into an enzymatically active dimer upon interaction with multiple DNA sites.

来自大肠杆菌K12 的Mrr是一种 IV 型限制性内切核酸酶，其作用是识别和切割外源甲基化 DNA。除了这种保护作用之外，Mrr 还可以造成染色体 DNA 损伤，在特定甲基转移酶（如 M.HhaII）异源表达时或在暴露于高压 (HP) 后引发宿主细胞中的 SOS 反应。响应这些扰动的 Mrr 激活涉及将无活性的同源四聚体解离为活性二聚体的寡聚开关。在这里，我们使用扫描数和亮度 (sN&B) 分析来确定体内组成型活性 Mrr 突变体的化学计量，预测为二聚体，并检查其他 GFP-Mrr 突变体对 M.HhaII 活性或 HP 休克的反应受损。我们还观察到在体外通过纯化的 GFP-Mrr 的 HP 荧光相关光谱法直接压力诱导四聚体解离。为了阐明亚基相互作用与 Mrr 及其变体的活性之间的联系，我们建立了与 DNA 结合的全长四聚体的结构模型。与功能相关的核酸内切酶类似，保守的 DNA 切割结构域将被 Mrr 无活性四聚体中的 DNA 识别结构域隔离，在与多个 DNA 位点相互作用时解离成具有酶活性的二聚体。