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Published 1984 | public
Journal Article

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 · Fe II) at 10^(-6) M concentration efficiently cleaves pBR322 DNA (10^(-5) M 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 gel analysis of the DNA cleavage patterns by ED • Fe (II) and DE • Fe (II) on both DNA strands of a 381 base pair restriction fragment reveals asymmetric DNA cleavage patterns. Cleavage is shifted to the 3' side 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 the 3' end of the thymine rich strand. 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.

Additional Information

© 1984 Elsevier Ltd. Received 1 October 1982. Supported by Fellowship DRG-526 of the Damon Runyon-Walter Winchell Cancer Fund.

Additional details

Created:
August 19, 2023
Modified:
October 18, 2023