Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA
Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA
复制标题
DOI：
10.1016/j.dnarep.2007.08.002
复制
发表时间：
2008-01-01
期刊：
影响因子：
3.8
通讯作者：
Diaz, Marilyn
中科院分区：
文献类型：
--
作者：
Brar, Sukhdev S.;Sacho, Elizabeth J.;Diaz, Marilyn
免疫球蛋白基因的高突变和类切换重组是胞苷脱氨酶AID触发的抗原激活机制。AID可使免疫球蛋白基因可变区和转换区DNA中的胞苷残基脱氨基。由此产生的尿嘧啶在高度突变的情况下诱导容易出错的DNA合成，或者在类开关重组的情况下激活非同源重组的DNA断裂。体外研究表明，AID可使单链底物而非双链底物脱氨化，除非AID与RPA形成复合体，且底物正在进行转录。然而，目前还不清楚AID主要是作为单体还是作为更高阶低聚物来脱氨基。为了单独检测AID的寡聚状态，以及在各种结构的单链DNA底物存在的情况下，包括嵌入双链DNA的环，我们使用原子力显微镜(AFM)单独或在与DNA的复合体中显示AID蛋白。令人惊讶的是，AFM结果表明，大多数AID分子以单体形式存在，并且在单链DNA底物发生有效脱胺的浓度下，它以单体的形式与单链DNA底物结合。在过量和限制蛋白的条件下，脱氨率也表明AID可以作为单体对单链底物进行脱胺。这些结果表明，在体外，非磷酸化的AID作为单体在单链DNA上具有催化活性，包括在环中发现的单链DNA，类似于转录过程中在免疫球蛋白切换区瞬时形成的单链DNA。(C)2007 Elsevier B.V.保留所有权利。
Hypermutation and class switch recombination of immunoglobulin genes are antigen-activated mechanisms triggered by AID, a cytidine deaminase. AID deaminates cytidine residues in the DNA of the variable and the switch regions of the immunoglobulin locus. The resulting uracil induces error-prone DNA synthesis in the case of hypermutation or DNA breaks that activate non-homologous recombination in the case of class switch recombination. In vitro studies have demonstrated that AID deaminates single-stranded but not double-stranded substrates unless AID is in a complex with RPA and the substrate is actively undergoing transcription. However, it is not clear whether AID deaminates its substrates primarily as a monomer or as a higher order oligomer. To examine the oligomerization state of AID alone and in the presence of single-stranded DNA substrates of various structures, including loops embedded in double-stranded DNA, we used atomic force microscopy (AFM) to visualize AID protein alone or in complex with DNA. Surprisingly, AFM results indicate that most AID molecules exist as a monomer and that it binds single-stranded DNA substrates as a monomer at concentrations where efficient deamination of single-stranded DNA substrates occur. The rate of deamination, under conditions of excess and limiting protein, also imply that AID can deaminate single-stranded substrates as a monomer. These results imply that non-phosphorylated AID is catalytically active as a monomer on single-stranded DNA in vitro, including single-stranded DNA found in loops similar to those transiently formed in the immunoglobulin switch regions during transcription. (C) 2007 Elsevier B.V. All rights reserved.