Larijani Mani, Petrov Alexander P, Kolenchenko Oxana, Berru Maribel, Krylov Sergey N, Martin Alberto
Department of Immunology, University of Toronto, Medical Sciences Bldg. 5265, Toronto, Ontario, Canada M5S 1A8.
Mol Cell Biol. 2007 Jan;27(1):20-30. doi: 10.1128/MCB.00824-06. Epub 2006 Oct 23.
Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and single-stranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors.
激活诱导的胞苷脱氨酶（AID）通过使单链DNA上的胞苷脱氨来启动二次抗体多样化过程。AID优先使位于W（A/T）R（A/G）二核苷酸之前的胞苷发生突变，这种序列特异性从硬骨鱼到人类在进化上是保守的。为了揭示AID的生化机制，我们比较了AID在WRC（一个热点基序，其中W等于A或T，R等于A或G）和非WRC基序上的催化和结合动力学。我们发现，尽管纯化的AID在体内观察到的相同程度上优先使WRC基序而非非WRC基序脱氨，但它对这两种基序表现出相似的结合亲和力，这表明其序列特异性并非由于WRC基序的优先结合。AID优先使五到七个核苷酸的气泡底物脱氨，而不是更大的气泡，并优先结合气泡型而非单链DNA底物，这表明AID的天然靶点要么是转录气泡，要么是茎环结构。重要的是，AID对单链DNA表现出非常高的亲和力，低解离常数和典型的转录因子及单链DNA结合蛋白的复合物解离长半衰期表明了这一点。这些发现表明，AID在脱氨后可能会持续存在于免疫球蛋白和其他靶序列上，可能作为一种支架蛋白来招募其他因子。
