Nucleotide preferences for DNA interstrand cross-linking induced by the cyclopropylpyrroloindole analogue U-77,779.

Abstract

Using a 21-base-pair duplex oligonucleotide containing a centrally located defined cross-linkable site, we have separated by gel electrophoresis DNA interstrand cross-links (ISC) from monofunctionally alkylated DNA (MA) and investigated the sequence selectivity for DNA ISC induced by the CC-1065 analogue U-77,779 (U-77). Sequencing gel analysis shows that U-77 induces two distinct types of DNA ISC. The first distinct form of DNA ISC spans six nucleotides and links two adenine N3 positions within an A/T-rich sequence. The second distinct DNA ISC spans seven nucleotides, also linking two adenine N3 positions, with a preference for contiguous runs of adenines. Three major 6-nucleotide DNA ISC's were identified and found to occur within 5'-TAATTA-3', 5'-TAAATA-3', and 5'-TAAAAA-3' sequences. The major 7-nucleotide DNA ISC was found to occur within 5'-TAAAAAA-3' sequences. Within this sequence, the formation of the 7-nucleotide DNA ISC was preferred over the 6-nucleotide DNA ISC by a ratio of approximately 2:1. DNA ISC formation within adenine tracts eliminated the inherent DNA bending associated with such sequences. Further, chemical probing of each isolated DNA ISC with diethyl pyrocarbonate (A-specific) and potassium permanganate (T-specific) shows that the major DNA conformational changes, such as helical distortion, were localized within the cross-linked sequence. These results suggest that a significant degree of DNA distortion may occur as a consequence of interstrand cross-linking.