DNA affinity cleaving : Sequence specific cleavage of DNA by Distamycin-EDTA - Fe(II) and EDTA-distamycin Fe(II)

Abstract

The attachment of EDTA· Fe(II) to distamycin changes the sequence specific DNA binding antibiotic into a sequence specific DNA cleaving molecule. We report the synthesis of EDTA-distamycin ( ED) which has the metal chelator, EDTA, tethered to the carboxy terminus of the N-methylpyrrole tripeptide moiety of the antiobiotic, distamycin. EDTA-distamycin- Fe(II) ( ED)· FeII at 10 -6M concentration efficiently cleaves pBR322 DNA (10 -5M in base pairs) in the presence of oxygen and dithiothreitol ( DTT). Using Maxam-Gilbert sequencing gel analyses, we find that ED· Fe(II) affords DNA cleavage patterns of unequal intensity covering two to four contiguous base pairs adjacent to a five base pair site consisting of adenines (A) and thymines (T). The multiple cleavages at each site might be evidence for a diffusible oxidizing species, perhaps hydroxyl radical. The unequal intensity of cleavage on each side of the A + T site permit assignment of major and minor orientations of the tripeptide binding unit. A comparison of the cleavage specificity of ED· Fe(II) with distamycin- EDTA· Fe(II), ( DE· Fe(II)) which has EDTA · Fe(II) attached to the amino terminus of the N-methylpyrrole tripeptide, shows DNA cleavage patterns at the same sites but with intensities of opposite polarity. Maxam-Gilbert sequencing el analysis of the DNA cleavage patterns by ED Fe(II) and DE Fe(II) on both DNA strands of a 381 se pair restriction fragment reveals asymmetric DNA cleavage patterns. Cleavage is shifted to the 3' de of each DNA strand. A model consistent with this cleavage pattern indicates one preferred binding te for ED Fe(II) and DE Fe(II) is 3'- TTTAA-5' with the “amino end” of the tripeptide oriented to e 3' end of the thymine rich strand. p]This “ DNA affinity cleavage” method which consists of attaching cleaving functions to DNA binding molecules followed by DNA cleavage pattern analyses using Maxam-Gilbert sequencing gels may be a useful direct method for determining the binding site and orientation of small molecules on native DNA.