Anti-cruciform monoclonal antibody and cruciform DNA interaction.
Cruciform DNA structure, as a structural feature, has been associated with regulation of transcription, recombination and replication. Previously used to successfully modify DNA replication and affinity-purify origins and autonomously replicating sequences. Using enzyme protection assays, their binding activity has been localized to the base (elbow) of the cruciform stem. We report here the hydroxyl radical footprinting of 2D3 (kappa IgG1) anti-cruciform monoclonal antibody on a stable cruciform structure created by heteroduplexing fragments from two plasmids, identical except for two centrally located palindromes of different sequence. The footprinting was performed at near-physiological salt concentrations, conditions favouring the stacked X-structure of the cruciform. Our data show that binding by the antibody occurs at the four-way junction (elbows) of the stable cruciform. The binding of the antibody seems also to cause associated structural distortions in the heteroduplex, which generally result in greater sensitivity to hydroxyl radicals at the tips of the cruciforms. The data are consistent to hydroxyl radicals at the tips of the cruciforms. The data are consistent with the binding of a single antibody to an antigen-combining site. The results of this study compare favourably with the hydroxyl radical footprinting studies reported recently for a human cruciform binding protein (CBP), which binds at the base of the stem-loop structure and causes similar distortions of the stable cruciform structure. These studies indicate that the four-way junction of the cruciform possesses certain unique structural qualities that are antigenic; the association of this structural determinant with DNA replication and the existence of a novel cellular protein, CBP, of similar binding specificity as the antibody specificity support a role for cruciforms as important regulatory recognition signals in replication.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018983:DNA Footprinting; D004261:DNA Replication; D000939:Epitopes; D006801:Humans; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D010957:Plasmids; D011506:Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D009692:Nucleic Acid Heteroduplexes; D011506:Proteins; D017665:Hydroxyl Radical; D004247:DNA