Department of Medicine, Raymond and Ruth Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Nat Chem Biol. 2012 Sep;8(9):751-8. doi: 10.1038/nchembio.1042. Epub 2012 Jul 8.
Activation-induced deaminase (AID)/APOBEC-family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, a major process for transcriptional activation. Although oxidation-dependent pathways for demethylation have been described, pathways involving deamination of either 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) have emerged as alternatives. Here we address the biochemical plausibility of deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on 5mC relative to cytosine, their canonical substrate, and no detectable deamination of 5hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of 5hmC was undetectable in genomic DNA, whereas oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation.
激活诱导的脱氨酶（AID）/APOBEC 家族胞嘧啶脱氨酶，已知在从抗体多样化到 mRNA 编辑的各种细胞过程中发挥作用，也与 DNA 去甲基化有关，这是转录激活的主要过程。尽管已经描述了依赖氧化的去甲基化途径，但涉及 5-甲基胞嘧啶（5mC）或 5-羟甲基胞嘧啶（5hmC）脱氨的途径已成为替代途径。在这里，我们探讨了脱氨偶联去甲基化的生化可行性。我们发现，与它们的典型底物胞嘧啶相比，纯化的 AID/APOBEC 对 5mC 的活性大大降低，并且对 5hmC 没有检测到脱氨作用。这一发现可以通过一系列修饰底物的反应性来解释，其中空间位阻对脱氨的不利影响越来越大。此外，在 AID/APOBEC 过表达后，5hmC 的脱氨产物在基因组 DNA 中无法检测到，而氧化中间体仍然可以检测到。我们的结果表明，胞嘧啶脱氨的空间位阻要求是脱氨酶在 DNA 去甲基化中提出的功能的内在障碍之一。
