Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, ‡Chair for Theoretical Chemistry at the Department of Chemistry, §Center for Drug Research at the Department of Pharmacy, Ludwig-Maximilians-Universität München , Butenandtstrasse 5-13, 81377 Munich, Germany.
J Am Chem Soc. 2013 Oct 2;135(39):14593-9. doi: 10.1021/ja403229y. Epub 2013 Sep 17.
Three new cytosine derived DNA modifications, 5-hydroxymethyl-2'-deoxycytidine (hmdC), 5-formyl-2'-deoxycytidine (fdC) and 5-carboxy-2'-deoxycytidine (cadC) were recently discovered in mammalian DNA, particularly in stem cell DNA. Their function is currently not clear, but it is assumed that in stem cells they might be intermediates of an active demethylation process. This process may involve base excision repair, C-C bond cleaving reactions or deamination of hmdC to 5-hydroxymethyl-2'-deoxyuridine (hmdU). Here we report chemical studies that enlighten the chemical reactivity of the new cytosine nucleobases. We investigated their sensitivity toward oxidation and deamination and we studied the C-C bond cleaving reactivity of hmdC, fdC, and cadC in the absence and presence of thiols as biologically relevant (organo)catalysts. We show that hmdC is in comparison to mdC rapidly oxidized to fdC already in the presence of air. In contrast, deamination reactions were found to occur only to a minor extent. The C-C bond cleavage reactions require the presence of high concentration of thiols and are acid catalyzed. While hmdC dehydroxymethylates very slowly, fdC and especially cadC react considerably faster to dC. Thiols are active site residues in many DNA modifiying enzymes indicating that such enzymes could play a role in an alternative active DNA demethylation mechanism via deformylation of fdC or decarboxylation of cadC. Quantum-chemical calculations support the catalytic influence of a thiol on the C-C bond cleavage.
三种新的胞嘧啶衍生 DNA 修饰物，5-羟甲基-2'-脱氧胞嘧啶（hmdC）、5-甲酰基-2'-脱氧胞嘧啶（fdC）和 5-羧基-2'-脱氧胞嘧啶（cadC）最近在哺乳动物 DNA 中被发现，特别是在干细胞 DNA 中。它们的功能目前尚不清楚，但据推测，在干细胞中，它们可能是一种活跃的去甲基化过程的中间产物。这个过程可能涉及碱基切除修复、C-C 键断裂反应或 hmdC 脱氨生成 5-羟甲基-2'-脱氧尿嘧啶（hmdU）。在这里，我们报告了一些化学研究，这些研究阐明了新胞嘧啶碱基的化学反应性。我们研究了它们对氧化和脱氨的敏感性，并研究了 hmdC、fdC 和 cadC 在不存在和存在生物相关（有机）催化剂巯基的情况下的 C-C 键断裂反应性。我们发现，与 mdC 相比，hmdC 在空气中存在的情况下很快被氧化为 fdC。相比之下，脱氨反应只发生在很小的程度上。C-C 键断裂反应需要高浓度的巯基存在，并受酸催化。虽然 hmdC 去羟甲基化非常缓慢，但 fdC 尤其是 cadC 与 dC 的反应速度要快得多。巯基是许多 DNA 修饰酶的活性部位残基，这表明这些酶可能在一种替代的活性 DNA 去甲基化机制中发挥作用，通过 fdC 的去甲酰化或 cadC 的脱羧化。量子化学计算支持了巯基对 C-C 键断裂的催化影响。
