Shlyakhtenko Luda S, Lushnikov Alexander J, Li Ming, Harris Reuben S, Lyubchenko Yuri L
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Center for Genome Engineering, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
PLoS One. 2014 Jun 6;9(6):e99354. doi: 10.1371/journal.pone.0099354. eCollection 2014.
The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.
载脂蛋白B mRNA编辑酶催化多肽样3（APOBEC3）家族的DNA胞嘧啶脱氨酶发挥作用，以阻止包括HIV-1在内的内源性逆转录元件和逆转录病毒的传播。其效力在家族成员中因寄生底物类型而异。APOBEC3A（A3A）在人类酶中是独特的，因为它主要在髓系谱系细胞类型中表达，被1型干扰素等先天免疫激动剂强烈诱导，并且能够容纳正常的和5-甲基胞嘧啶核碱基。在这里，我们应用原子力显微镜（AFM），采用一种杂交DNA方法来表征A3A与单链和双链DNA之间的相互作用，在这种方法中，单链区域两侧是确定长度的双链体。AFM图像分析揭示了A3A与单链DNA的结合，并且这种相互作用在高蛋白与DNA比例（至少100∶1）时最为明显（复合物产率约80%）。A3A与单链DNA结合时主要是单体形式，并且在高达50 nM的浓度下在溶液中也是单体形式。这些特性与最近的生物化学、生物物理和结构研究结果非常吻合。然而，这些特征与相关酶APOBEC3G的特征形成对比，在类似的测定中，APOBEC3G可以以单体形式存在，但倾向于以浓度依赖的方式形成寡聚体。这些AFM数据表明，A3A具有将其与APOBEC3G区分开来的内在生物物理差异。本文讨论了这些特性与先天免疫中生物学功能之间的潜在关系。
