Enhancement of Oxidative Cleavage of DNA by the Binding Sites of Two Anti-double-stranded DNA Antibodies

Tetsuo Kubota,Naomi Watanabe,Yoshiyuki Kanai,B David Stollar

doi:10.1074/jbc.271.11.6555

Abstract

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.

Highlights

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
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
In competitive immunoassays with a series of synthetic double-stranded oligonucleotides, H241 bound to a3 or4 core in the center of a base-paired octadecanucleotide [21]

Summary

Introduction

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. The enhanced cleavage occurred preferentially at dA-dTrich 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. DNA-binding sites of monoclonal anti-DNA antibodies and epitopes have been mapped by competitive immunoassay with polynucleotides or oligonucleotides [17, 18]. In competitive immunoassays with a series of synthetic double-stranded oligonucleotides, H241 bound to a (dG-dC) or (dG-dC) core in the center of a base-paired octadecanucleotide [21] This selectivity was reflected in the very marked preference of this antibody for poly(dG-dC) over poly(dA-dT). We found that these two anti-dsDNA antibodies enhanced rather than protected against the cleavage of DNA by hydroxyl radical-generating systems. The two antibodies differed in the conditions under which they were effective and in the sites of DNA cleavage they supported

Methods

Results

Conclusion