Martomo Stella A, Fu Dongtao, Yang William W, Joshi Nikhil S, Gearhart Patricia J
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
J Immunol. 2005 Jun 15;174(12):7787-91. doi: 10.4049/jimmunol.174.12.7787.
Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and class switch recombination of Ig genes in B cells. Although AID has been shown to deaminate deoxycytidine to deoxyuridine in DNA in vitro, there is no physical evidence for increased uracils in DNA from cells expressing AID in vivo. We used several techniques to detect uracil bases in a gene that was actively transcribed in Escherichia coli cells expressing AID. Plasmid DNA containing the gene was digested with uracil-DNA glycosylase to remove uracil, and apurinic/apryimidinic endonuclease to nick the abasic site. The nicked DNA was first analyzed using alkaline gel electrophoresis, in which there was a 2-fold increase in the linear form of the plasmid after AID induction compared with plasmid from noninduced bacteria. Second, using a quantitative denaturing Southern blot technique, the gene was predominantly nicked in the nontranscribed strand compared with the transcribed strand. Third, using ligation-mediated PCR, the nicks were mapped on the nontranscribed strand and were located primarily at cytosine bases. These data present direct evidence for the presence of uracils in DNA from cells that are induced to express AID, and they are preferentially generated at cytosines in the nontranscribed strand during transcription.
激活诱导的胞苷脱氨酶（AID）是B细胞中Ig基因的体细胞超突变和类别转换重组所必需的。尽管在体外实验中已证明AID可使DNA中的脱氧胞苷脱氨生成脱氧尿苷，但尚无体内表达AID的细胞DNA中尿嘧啶增加的直接证据。我们使用了多种技术来检测在表达AID的大肠杆菌细胞中活跃转录的基因中的尿嘧啶碱基。含有该基因的质粒DNA先用尿嘧啶-DNA糖基化酶消化以去除尿嘧啶，再用脱嘌呤/脱嘧啶内切酶在无碱基位点处切口。首先使用碱性凝胶电泳分析切口后的DNA，结果显示与未诱导细菌的质粒相比，诱导表达AID后质粒的线性形式增加了2倍。其次，使用定量变性Southern印迹技术，与转录链相比，该基因在非转录链上主要出现切口。第三，使用连接介导的PCR，将切口定位在非转录链上，且主要位于胞嘧啶碱基处。这些数据直接证明了诱导表达AID的细胞DNA中存在尿嘧啶，并且在转录过程中它们优先在非转录链的胞嘧啶处产生。
