Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
Nucleic Acids Res. 2012 Oct;40(18):9206-17. doi: 10.1093/nar/gks685. Epub 2012 Jul 13.
The AID/APOBEC family of enzymes in higher vertebrates converts cytosines in DNA or RNA to uracil. They play a role in antibody maturation and innate immunity against viruses, and have also been implicated in the demethylation of DNA during early embryogenesis. This is based in part on reported ability of activation-induced deaminase (AID) to deaminate 5-methylcytosines (5mC) to thymine. We have reexamined this possibility for AID and two members of human APOBEC3 family using a novel genetic system in Escherichia coli. Our results show that while all three genes show strong ability to convert C to U, only APOBEC3A is an efficient deaminator of 5mC. To confirm this, APOBEC3A was purified partially and used in an in vitro deamination assay. We found that APOBEC3A can deaminate 5mC efficiently and this activity is comparable to its C to U deamination activity. When the DNA-binding segment of AID was replaced with the corresponding segment from APOBEC3A, the resulting hybrid had much higher ability to convert 5mC to T in the genetic assay. These and other results suggest that the human AID deaminates 5mC's only weakly because the 5-methyl group fits poorly in its DNA-binding pocket.
高等脊椎动物中的 AID/APOBEC 酶家族将 DNA 或 RNA 中的胞嘧啶转化为尿嘧啶。它们在抗体成熟和先天免疫病毒中发挥作用，并且在胚胎早期的 DNA 去甲基化中也被牵连。这部分是基于报道的激活诱导脱氨酶 (AID) 将 5-甲基胞嘧啶 (5mC) 脱氨为胸腺嘧啶的能力。我们使用大肠杆菌中的新型遗传系统重新检查了 AID 和人 APOBEC3 家族的两个成员的这种可能性。我们的结果表明，虽然所有三个基因都显示出将 C 转化为 U 的强大能力，但只有 APOBEC3A 是 5mC 的有效脱氨酶。为了证实这一点，我们部分纯化了 APOBEC3A 并将其用于体外脱氨测定。我们发现 APOBEC3A 可以有效地脱氨 5mC，其活性与 C 至 U 的脱氨活性相当。当 AID 的 DNA 结合片段被 APOBEC3A 的相应片段取代时，所得的杂种在遗传测定中具有更高的将 5mC 转化为 T 的能力。这些和其他结果表明，人类 AID 对 5mC 的脱氨作用很弱，因为 5-甲基基团在其 DNA 结合口袋中不太合适。
