Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

抗病毒 DNA 胞嘧啶脱氨酶 APOBEC3A 和 APOBEC3B 是催化胞嘧啶-尿嘧啶脱氨作用的癌症突变的主要来源。APOBEC3A 优先靶向单链 DNA，对采用茎环二级结构的 DNA 区域具有显著的亲和力。然而，APOBEC3A 和 APOBEC3B 的详细底物偏好尚未完全确定，DNA 序列对 APOBEC3A 和 APOBEC3B 脱氨酶活性的特定影响仍有待研究。在这里，我们发现 APOBEC3B 还选择性地靶向 DNA 茎环结构，它们与那些被 APOBEC3A 脱氨的 DNA 不同。我们开发了 Oligo-seq，这是一种基于体外测序的方法，用于识别促进 APOBEC3A 和 APOBEC3B 活性的特定序列上下文。通过这种方法，我们证明了 APOBEC3A 和 APOBEC3B 脱氨酶活性受到靶向胞嘧啶周围特定序列的强烈调节。此外，我们确定了APOBEC3B的结构特征和负责其基材偏好的APOBEC3A。重要的是，我们确定肿瘤基因组内发夹形成序列的 APOBEC3B 诱导突变与APOBEC3A突变的 DNA 茎环序列不同。总之，我们的研究提供了证据，证明 APOBEC3A 和 APOBEC3B 可以在癌症基因组中产生不同的突变景观，这由它们独特的底物选择性驱动。