Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA.
Nucleic Acids Res. 2013 Jul;41(12):6139-48. doi: 10.1093/nar/gkt246. Epub 2013 Apr 24.
Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are 'hotspots' for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3' and 5' of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.
载脂蛋白 B mRNA 编辑酶催化多肽 3G（即 APOBEC3G 或 A3G）是一种进化上保守的胞嘧啶脱氨酶，可有效限制人类免疫缺陷病毒 1 型（HIV-1）、逆转录转座子和其他病毒。A3G 对胞嘧啶二核苷酸具有核苷酸靶位特异性，但只有某些胞嘧啶二核苷酸是胞嘧啶脱氨酶的“热点”，而其他胞嘧啶二核苷酸则很少或几乎不受 A3G 的编辑。定义这些关键 A3G 热点的因素尚未完全了解。为了研究 A3G 热点是如何定义的，我们使用体外荧光共振能量转移基于寡核苷酸测定法来探测 A3G 的靶位特异性。我们的研究结果强烈表明，靶单链 DNA（ssDNA）二级结构以及直接位于胞嘧啶二核苷酸 3' 和 5' 的碱基是 A3G 识别的关键因素。例如，A3G 不易脱氨 ssDNA 茎结构中的胞嘧啶二核苷酸或由三个碱基组成的核苷酸碱基环中的胞嘧啶二核苷酸。除非胞嘧啶残基侧翼有胞嘧啶二核苷酸，否则长达七个碱基的单链核苷酸环不是 A3G 活性的良好靶标。此外，我们观察到 A3G 倾向于在 ssDNA 无规卷曲区侧翼的胞嘧啶二核苷酸靶标周围的腺嘌呤、胞嘧啶和胸腺嘧啶。当鸟嘌呤侧翼有胞嘧啶二核苷酸时，检测到低的胞嘧啶脱氨酶活性。总之，我们的研究结果首次证明 A3G 胞嘧啶脱氨热点是由胞嘧啶二核苷酸靶标的序列上下文以及 ssDNA 二级结构共同定义的。这些知识可用于更好地追踪 A3G 活性突变的起源，并阐明其对 HIV-1 遗传变异等过程的影响。
