Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
Nucleic Acids Res. 2020 Nov 18;48(20):e118. doi: 10.1093/nar/gkaa845.
The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of this family, APOBEC3A (A3A) and APOBEC3B, deaminate cytosines in the lagging-strand template during replication. To obtain direct evidence for the presence of these uracils, we engineered a protein that covalently links to DNA at uracils, UdgX, for mammalian expression and immunohistochemistry. We show that UdgX strongly prefers uracils in ssDNA over those in U•G or U:A pairs, and localizes to nuclei in a dispersed form. When A3A is expressed in these cells, UdgX tends to form foci. The treatment of cells with cisplatin, which blocks replication, causes a significant increase in UdgX foci. Furthermore, this protein- and hence the uracils created by A3A- colocalize with replication protein A (RPA), but not with A3A. Using purified proteins, we confirm that RPA inhibits A3A by binding ssDNA, but despite its overexpression following cisplatin treatment, RPA is unable to fully protect ssDNA created by cisplatin adducts. This suggests that cisplatin treatment of cells expressing APOBEC3A should cause accumulation of APOBEC signature mutations.
AID/APOBEC 酶使单链 DNA（ssDNA）中的胞嘧啶脱氨，在先天和适应性免疫中发挥关键作用。由此产生的尿嘧啶导致突变和链断裂，从而使病毒失活并使抗体库多样化。突变证据表明，该家族的两个成员 APOBEC3A（A3A）和 APOBEC3B 在复制过程中使滞后链模板中的胞嘧啶脱氨。为了获得这些尿嘧啶存在的直接证据，我们设计了一种可将 DNA 与尿嘧啶共价连接的蛋白质，即用于哺乳动物表达和免疫组化的 UdgX。我们表明，UdgX 强烈偏好 ssDNA 中的尿嘧啶，而不是 U•G 或 U:A 对中的尿嘧啶，并且以弥散形式定位于核内。当 A3A 在这些细胞中表达时，UdgX 往往会形成焦点。用顺铂处理细胞会阻止复制，从而导致 UdgX 焦点显著增加。此外，这种蛋白质 - 以及由 A3A 产生的尿嘧啶 - 与复制蛋白 A（RPA）共定位，但与 A3A 不共定位。使用纯化的蛋白质，我们证实 RPA 通过结合 ssDNA 抑制 A3A，但尽管顺铂处理后其过表达，RPA 仍无法完全保护由顺铂加合物产生的 ssDNA。这表明表达 APOBEC3A 的细胞经顺铂处理后应会积累 APOBEC 特征性突变。
