Molecular basis for sequence-specific DNA alkylation by CC-1065

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Abstract
CC-1065 is a potent antitumor antibiotic that binds covalently to N3 of adenine in the minor groove of DNA. The CC-1065 molecule is made up of three repeating pyrroloindole subunits, one of which (the left-hand one or A subunit) contains a reactive cyclopropyl function. The drug reacts with adenines in DNA in a highly sequence-specific manner, overlapping four base pairs to the 5'' side of the covalently modified base. Concomitant with CC-1065 covalent binding to DNA is an asymmetric effect on local DNA structure which extends more than one helix turn to the 5''-side of the covalent binding site. The DNA alkylation, sequence specificity, and biological potency of CC-1065 and a select group of trimeric synthetic analogues were evaluated. The results suggest that (a) noncovalent interactions between this series of compounds and DNA do not lead to the formation of complexes stable enough to be detected by footprinting methods, (b) sequence specificity and alkylation intensity can be modulated by the substituents on the nonreactive middle and right-hand segments and (c) biological potency correlates well with ability to alkylate DNA. In addition, the extent and the sequence specificity of covalent adduct formation between linear DNA fragments and three analogues comprised of the CC-1065 alkylating subunit linked to zero (analogue A), one (analogue AB), or two (analogue ABC) nonreactive indole subunits were compared. The results suggest that specificity of covalent binding of this analogue series is controlled not by the noncovalent interactions of the B and C subunits with the minor groove but by sequence-dependent reactivity of adenines with the alkylating (A) subuint. However, the B and C subunits markedly increase the apparent rate constant of the reaction with "susceptible'' adenines, suggesting that these moieties facilitate noncovalent interactions preceding covalent bond formation. Covalent binding of the analogue consisting only of the alkylating subunit of CC-1065 (analogue A) was associated with the same large asymmetric effect on DNA structure as the entire CC-1065 molecule. This altered local DNA structure could be a consequence of adduct formation. Alternatively, it may be indirect evidence of a particular DNA conformation which existed prior to covalent bond formation and which was "trapped by the drug. It is proposed that certain adenine-containing sequences have an increased propensity to undergo such a local conformational change and that this is the molecular basis for sequence specificy of these DNA-reactive compounds. The results provide strong experimental evidence for the importance of sequence-dependent site reactivity, rather than noncovalent minor groove interactions, in determining the alkylation specificity of some DNA-reactive molecules.