Veterinary Medicine Graduate Program, University of Minnesota, Minneapolis, MN 55455 USA.
Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455 USA.
Nucleic Acids Res. 2020 Feb 20;48(3):1353-1371. doi: 10.1093/nar/gkz1164.
The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5'TC3' or 5'CT3' dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.
人类载脂蛋白 B mRNA 编辑酶、催化多肽样 3（APOBEC3，A3）家族成员蛋白可以使单链（ss）DNA 中的胞嘧啶脱氨基，这限制了人类免疫缺陷病毒 1 型（HIV-1）、反转录转座子和其他病毒，如乙型肝炎病毒，但可以在许多癌症中引起诱变表型。虽然有几种 A3 蛋白的结构信息，但脱氨酶靶标选择的确切细节尚不完全清楚。在这里，我们报告了首次使用六种纯化的 A3 成员酶、具有 5'TC3'或 5'CT3'二核苷酸靶位点的寡核苷酸以及不同二级结构中的不同侧翼碱基，对 A3 脱氨作用的靶位选择进行平行、比较分析。观察到 A3A、A3F 和 A3H 对侧翼为 A 或 T 的 TC 靶标有强烈的偏好，而所有检查的 A3 蛋白都不偏爱侧翼为 G 的 TC 靶标。我们观察到 TC 靶标在 ssDNA 区域比 dsDNA、环或凸起区域更强烈地被优先选择，侧翼碱基影响偏好程度。CT 也被证明是一个潜在的脱氨作用靶标。总之，我们的观察结果为 A3 酶靶标选择以及 A3 诱变如何影响突变率提供了新的见解。
