Ultraviolet cross-linking of helical oligonucleotides to two monoclonal MRL-1pr/1pr anti-DNA autoantibodies. Variations in H and L chain binding to DNA.
Experiments were performed to determine whether both H and L chains of different anti-native DNA autoantibodies are uniformly involved in binding to DNA. Two purified monoclonal mouse (MRL-1pr/1pr) IgG autoantibodies, H241 and 2C10, were tested. They both bound synthetic helical oligonucleotides of 10 to 20 base pairs in a gel electrophoresis retardation assay but differed in their preferences for given base sequences. Exposure of antibody-radiolabeled oligonucleotide mixtures to UV light (254 nm) for 10 min led to specific covalent cross-linking of oligonucleotide to both the H and the L chains of H241 but only to the H chain of 2C10. Single labeling events were detected without higher aggregation. The oligonucleotides were not cross-linked to unrelated IgG, even after 2 h of irradiation. Cross-linked (radioactively labeled) H and L chains of H241 and 2C10 were isolated from denaturing electrophoresis gels and digested with lysyl endopeptidase and/or staphylococcal V8 protease. H241 and 2C10 H chains each yielded a major labeled peptide fragment, but the peptides from the two antibodies were different. These experiments measured only some of the antibody-DNA interactions, probably with bases in the major groove of the DNA. They indicated that two MRL-1pr/1pr IgG anti-native DNA antibodies differ in their H and L chain contacts with DNA and provide an approach to identifying affinity-labeled binding sites in the antibodies.
Jang
Y J
YJ
Department of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111.
Stollar
B D
BD
D000818:Animals; D000974:Antibodies, Antinuclear / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D008232:Lymphoproliferative Disorders / Q000276:immunology*; D051379:Mice; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009841:Oligonucleotides; D004247:DNA