(+)-
CC-1065 is a potent antitumor
antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-
CC-1065 are due to the ability of this compound to covalently modify
DNA. (+)-
CC-1065 reacts with duplex
DNA to form a (N3-adenine)-DNA adduct which lies in the minor groove of
DNA overlapping with a five base-pair region. As a consequence of covalent modification with (+)-
CC-1065, the helix
bends into the minor groove and also undergoes winding and stiffening. In the studies described here, we have constructed templates for helicase-catalyzed unwinding of
DNA that contain site-directed (+)-
CC-1065 and analogue
DNA adducts. Using these templates we have shown that (+)-
CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all produce a significant inhibition of unwinding of a 3'-tailed oligomer duplex by helicase II when the displaced strand is covalently modified. However, the extent of helicase II inhibition is much more significant for (+)-
CC-1065 and an analogue which also produced
DNA winding when the winding effects are transmitted in the opposite direction to the helicase unwinding activity. This observed pattern of inhibition of helicase-catalyzed unwinding of
drug-modified templates was the same for a 3'-T-tail, for different duplex region sequences, and with the Escherichia coli rep
protein. Unexpectedly, the gel mobility of the displaced
drug-modified single strand was dependent on the species of
drug attached to the
DNA. Last, strand displacement by helicase II coupled to primer extension by E. coli
DNA polymerase I showed the same pattern of inhibition when the lagging strand was covalently modified. In addition, the presence of helicase II on single-stranded regions of templates caused the premature termination of primer extension by
DNA polymerase. These results are discussed from the perspective that (+)-
CC-1065 and its analogues have different effects on
DNA structure, and these resulting structural changes in
DNA molecules are related to the different in vivo
biological consequences caused by these
drug molecules.