Characterization of the binding specificity of two anticruciform DNA monoclonal antibodies

Two monoclonal antibodies (2D3 and 4B4) have been raised against a stable cruciform DNA structure containing the 27-base pair palindrome of the SV40 origin of replication on one strand and an unrelated 26-base pair palindrome on the complementary strand (pRGM 21 x pRGM 29) and have been shown to recognize conformational determinants specific to cruciform DNA structures (Frappier, L., Price, G.B., Martin, R. G., and Zannis-Hadjopoulos, M. (1987) J. Mol. Biol. 193, 751-758). To define the region(s) of the cruciform that is recognized by these antibodies, we examined the ability of 2D3 and 4B4 to protect the single-stranded tips of the loops or the four-way junctions at the base of the stem of stable cruciform molecules against cleavage by mung bean nuclease or T7 endonuclease 3, respectively. Both antibodies were found to protect two of the four elbow-like structures at the base of the cruciform from T7 endonuclease 3 cleavage, but not the tips of the cruciform arms from mung bean nuclease cleavage. Also, predigestion of the cruciform with mung bean nuclease did not affect the binding of either antibody. In addition, 2D3 bound to a cruciform and a T-shaped structure involving the palindromic sequence at the cloning site of pUC7, which is completely unrelated in sequence to the palindrome of pRGM 21 x pRGM 29, and protected the base of these stem-loop structures against cleavage by T4 endonuclease VII. These results indicate that 2D3 and 4B4 bind at or near the base of the cruciform molecules and that, at least for 2D3, binding is independent of DNA sequence.