Inhibition of T4 DNA ligase activity by (+)-CC-1065: demonstration of the importance of the stiffening and winding effects of (+)-CC-1065 on DNA.
Non-denaturing gel electrophoresis analysis demonstrates that the stiffening and winding effects of (+)-CC-1065 produce unusual proximal and distal inhibition of T4 DNA ligase-catalysed ligation of covalently modified DNA. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds through N3 of adenine in DNA and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Previous studies (Lee et al., 1991) have shown that (+)-CC-1065 produces bending and winding of DNA. The DNA bending and sequence specificity is mediated by the alkylating 'A' subunit of (+)-CC-1065, while the close van der Waals contacts between the non-alkylating B and C subunits of (+)-CC-1065 and the floor of the minor groove of DNA are responsible for the winding of DNA. Covalent modification of oligomers with (+)-CC-1065 and structurally related drugs leads to preferential inhibition of T4 DNA ligase on the non-covalently modified strand to the 5' side of the covalent adduct site, but enhanced ligation of the covalently modified strand. We speculate that the differential effect on proximal strand ligation is due to a drug-induced winding and helix-stabilizing effect which occurs predominantly to the 5' side of the adduct. In addition to the proximal inhibition of ligation, we also describe a distal inhibition of T4 DNA ligase activity which occurs exclusively with drug-modified oligomers and that, if successful, would result in 180 degrees out-of-phase bent DNA following ligation. In this case, two 25 mers or a 21 plus a 29 mer are inhibited from ligation when modified with (+)-CC-1065. This distal ligation is unique to (+)-CC-1065 and its analogs that cause stiffening of the DNA helix. The (+)-CC-1065-induced stiffening effect was demonstrated using a circularization assay and was found to be associated with the close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove, and also to the benzofuran moiety of (+)-ABC" (Adozelesin), a (+)-CC-1065 analog. We conclude from these studies that the DNA-winding and helix-stabilizing effects of these drug molecules can dramatically affect the efficiency of proximal ligation mediated by T4 DNA ligase, and the unusual helix-stiffening effect of (+)-CC-1065, (+)-AB'C' and (+)-ABC" can stabilize the structure of bent DNA formed by drug modification, which results in distal ligase inhibition.
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Drug Dynamics Institute, College of Pharmacy, University of Texas, Austin 78712.
Hurley
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D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000187:drug effects*; D011088:DNA Ligases / Q000037:antagonists & inhibitors*; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins / Q000737:chemistry / Q000494:pharmacology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013329:Structure-Activity Relationship
D000903:Antibiotics, Antineoplastic; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins; C018782:CC 1065; D004247:DNA; D011088:DNA Ligases