DNA 甲基转移酶是骨髓增生异常综合征 (MDS)、慢性粒单核细胞白血病 (CMML)、急性髓性白血病 (AML) 以及可能的 β-血红蛋白病的药物靶标。我们描述了 DNA 中核苷类似物与原核 CpG 特异性 DNA 甲基转移酶 (M.MpeI) 的相互作用，作为哺乳动物 DNMT1 甲基转移酶的模型。我们测试了含有 5-羟甲基胞嘧啶 (5hmC)、5-羟基胞嘧啶 (5OHC)、5-甲基-2-嘧啶酮（核糖基化形式称为 5-methylzebularine, 5mZ）、5,6-二氢-5-氮杂胞嘧啶 (dhaC) 的 DNA )、5-氟胞嘧啶 (5FC)、5-氯胞嘧啶 (5ClC)、5-溴胞嘧啶 (5BrC) 和 5-碘胞嘧啶 (5IC)。迄今为止，5FC 的共价复合物形成效率最高。 dhaC 和 5mZ 的非共价复合物最为丰富。令人惊讶的是，我们观察到 5IC 和 5BrC 的甲基化，以及较小程度的 5ClC 和 5FC 在小分子硫醇亲核试剂存在下的甲基化，但在小分子硫醇亲核试剂不存在的情况下则没有。对于 5IC 和 5BrC，我们通过质谱证明反应是由于甲基转移酶驱动的脱卤作用，然后是甲基化作用。 M.MpeI-DNA 复合物的晶体结构捕获具有小或可旋转 (5mZ) 5-取代基的类似物的活性位点环的“内”构象，以及大体积 5-取代基的“外”构象。由于 DNMT1 也观察到非常相似的“内”和“外”环构象，因此我们的结论很可能推广到其他 DNA 甲基转移酶。© 作者 2024。由牛津大学出版社代表 Nucleic Acids Research 出版。

DNA methyltransferases are drug targets for myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myelogenous leukemia (AML) and possibly β-hemoglobinopathies. We characterize the interaction of nucleoside analogues in DNA with a prokaryotic CpG-specific DNA methyltransferase (M.MpeI) as a model for mammalian DNMT1 methyltransferases. We tested DNA containing 5-hydroxymethylcytosine (5hmC), 5-hydroxycytosine (5OHC), 5-methyl-2-pyrimidinone (in the ribosylated form known as 5-methylzebularine, 5mZ), 5,6-dihydro-5-azacytosine (dhaC), 5-fluorocytosine (5FC), 5-chlorocytosine (5ClC), 5-bromocytosine (5BrC) and 5-iodocytosine (5IC). Covalent complex formation was by far most efficient for 5FC. Non-covalent complexes were most abundant for dhaC and 5mZ. Surprisingly, we observed methylation of 5IC and 5BrC, and to a lesser extent 5ClC and 5FC, in the presence, but not the absence of small molecule thiol nucleophiles. For 5IC and 5BrC, we demonstrated by mass spectrometry that the reactions were due to methyltransferase driven dehalogenation, followed by methylation. Crystal structures of M.MpeI-DNA complexes capture the 'in' conformation of the active site loop for analogues with small or rotatable (5mZ) 5-substituents and its 'out' form for bulky 5-substituents. Since very similar 'in' and 'out' loop conformations were also observed for DNMT1, it is likely that our conclusions generalize to other DNA methyltransferases.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.