Kubota T, Watanabe N, Kanai Y, Stollar B D
School of Allied Health Sciences, Faculty of Medicine, Tokyo Medical and Dental University, Japan.
J Biol Chem. 1996 Mar 15;271(11):6555-61. doi: 10.1074/jbc.271.11.6555.
Nucleic acid specificity was tested for two monoclonal anti-double-stranded DNA autoantibodies, 2C10 and H241, derived from two lupus-prone MRL/Mp-lpr/lpr mice. Antibody 2C10 bound double-stranded oligonucleotides with a preference for dA-dT over dG-dC base pairs and did not bind single-stranded oligonucleotides. Distamycin A, an antibiotic that binds to the minor groove, inhibited 2C10 binding of double-stranded DNA, suggesting that this antibody interacts with dA-dT base pairs in the minor groove. Antibody H241 binding was previously shown to have a dG-dC preference and to involve both major and minor grooves. In attempted footprinting assays, both 2C10 and H241 markedly en- hanced rather than protected against cleavage of DNA by hydroxyl radical-generating systems. With 2C10, this enhancement effect was observed only when hydroxyl radical generation was associated with oxidation of Fe(II). In contrast, H241 enhancement occurred in the presence of H2O2 and ascorbate or UV light irradiation and did not depend on added metal ion. The enhancement sites were related to the antibody binding specificities. The oligonucleotide 5'-AAAATATATATTT-3' was a much more effective inhibitor of the 2C10 enhancement than of the H241 effect, whereas the oligonucleotide 5'-GGGGCGCGCGCCC-3' was a much more effective inhibitor of the H241 enhancement. In addition, the enhanced cleavage occurred preferentially at dA-dT-rich regions with 2C10 and at dG-dC-rich regions with H241. These findings raise the possibility that anti-DNA autoantibodies could enhance DNA damage in inflammatory lesions in which hydroxyl radicals are generated.
对源自两只狼疮易感MRL/Mp-lpr/lpr小鼠的两种单克隆抗双链DNA自身抗体2C10和H241进行了核酸特异性测试。抗体2C10结合双链寡核苷酸，对dA-dT碱基对的偏好高于dG-dC碱基对，且不结合单链寡核苷酸。与小沟结合的抗生素Distamycin A抑制2C10与双链DNA的结合，表明该抗体与小沟中的dA-dT碱基对相互作用。先前已证明抗体H241的结合具有dG-dC偏好，且涉及大沟和小沟。在尝试进行足迹分析时，2C10和H241均显著增强而非保护DNA免受产生羟基自由基的系统的切割。对于2C10，仅当羟基自由基的产生与Fe(II)的氧化相关时才观察到这种增强作用。相比之下，H241的增强作用在存在H2O2和抗坏血酸或紫外线照射的情况下发生，且不依赖于添加的金属离子。增强位点与抗体结合特异性相关。寡核苷酸5'-AAAATATATATTT-3'对2C10增强作用的抑制比对H241作用的抑制更有效，而寡核苷酸5'-GGGGCGCGCGCCC-3'对H241增强作用的抑制更有效。此外，2C10的增强切割优先发生在富含dA-dT的区域，而H241的增强切割优先发生在富含dG-dC的区域。这些发现增加了抗DNA自身抗体可能在产生羟基自由基的炎症病变中增强DNA损伤的可能性。
