Recombinant Phabs reactive with 7,8-dihydro-8-oxoguanine, a major oxidative DNA lesion.
Recombinant Phabs reactive with 7,8-dihydro-8-oxoguanine, a major oxidative DNA lesion.
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重组 Phab 与 7,8-二氢-8-氧代鸟嘌呤（一种主要的氧化 DNA 损伤）发生反应。
DOI：
10.1021/bi9517244
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
R. Melamede
中科院分区：
文献类型：
--
作者：
I. Bespalov;A. Purmal;M. Glackin;S. Wallace;R. Melamede
与 DNA 损伤结合的抗体 Fab 为了解 DNA 损伤特异性蛋白质相互作用提供了有用的模型。使用氧化 DNA 损伤的核苷和核苷酸衍生物 7,8-二氢-8-氧代鸟嘌呤 (8-oxoG) 和 7,8-二氢-8-氧代腺嘌呤 (8-oxoA) 的 BSA 和 RSA 缀合物来免疫小鼠。分离来自应答者的 RNA 并用于库克隆和噬菌体展示与这些半抗原结合的 Fab。直接结合和竞争性酶联免疫吸附测定 (ELISA) 表明，产生了对 8-氧代嘌呤-BSA 缀合物和 8-氧代鸟嘌呤具有特异性的噬菌体 Fab (Phabs)，尽管 Phabs 不与 DNA 中的 8-氧代嘌呤发生反应。具有不同结合特性的克隆之间的氨基酸序列比较表明，由互补决定区（CDR）限定的结合表面的相对小部分解释了半抗原结合特异性，而其他区域似乎通过与蛋白质或DNA表位相互作用来稳定半抗原结合。 8-氧代嘌呤-BSA结合Fab和DNA结合Fab之间的链改组表明，DNA结合剂的重链仅赋予8-氧代嘌呤-BSA结合剂之一的轻链DNA结合能力。 8-oxoG 特异性克隆 g37 的同源建模显示与先前分离的两种单链核酸特异性单克隆抗体显着相似。在不与 DNA 结合的 8-oxoG Fab 中，假定的 DNA 结合峡谷被 CDR2 区域中的重链残基阻断，并且由于轻链框架区的位置不同，似乎缺少部分峡谷壁。
Antibody Fabs that bind to DNA damages provide useful models for understanding DNA damage-specific protein interactions. BSA and RSA conjugates of the nucleoside and nucleotide derivatives of the oxidative DNA lesions, 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), were used to immunize mice. RNA from the responders was isolated and used to repertoire clone and phage display Fabs that bind to these haptens. Direct binding and competitive enzyme-linked immunosorbent assay (ELISA) demonstrated that phage Fabs (Phabs) specific for 8-oxopurine-BSA conjugates and 8-oxoguanine were produced although the Phabs did not react with 8-oxopurines in DNA. Amino acid sequence comparisons among clones having different binding properties suggested that a relatively small portion of the binding surfaces defined by the complementarity determining regions (CDR) accounted for hapten binding specificity, whereas other regions appeared to stabilize hapten binding by interacting with protein or DNA epitopes. Chain shuffling between 8-oxopurine-BSA binding Fabs and a DNA binding Fab showed that the heavy chain of the DNA binder conferred DNA binding capacity to the light chain of only one of the 8-oxopurine-BSA binders. Homology modeling of the 8-oxoG-specific clone g37 showed significant similarities to two previously isolated monoclonal antibodies specific for single-stranded nucleic acids. In the 8-oxoG Fab, which did not bind to DNA, the presumptive DNA binding canyon was blocked by heavy chain residues in the CDR2 region and appeared to lack part of the canyon wall due to the different placement of the light chain framework region.