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. Previ ously, anti-cruciform DNA specific monoclonal antibodies were prepared and used to successfully modify DNA replication and affinity-purify o rigins 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 footprinti ng of 2D3 (kappa IgG1) anti-cruciform monoclonal antibody on a stable cruciform structure created by heteroduplexing fragments from two plas mids, identical except for two centrally located palindromes of differ ent sequence. The footprinting was performed at near-physiological sal t concentrations, conditions favouring the stacked X-structure of the cruciform. Our data show that binding by the antibody occurs at the fo ur-way junction (elbows) of the stable cruciform. The binding of the a ntibody seems also to cause associated structural distortions in the h eteroduplex, which generally result in greater sensitivity 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 res ults of this study compare favourably with the hydroxyl radical footpr inting 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 i ndicate that the four-way junction of the cruciform possesses certain unique structural qualities that are antigenic; the association of thi s structural determinant with DNA replication and the existence of a n ovel cellular protein, CBP, of similar binding specificity as the anti body specificity support a role for cruciforms as important regulatory recognition signals in replication.