Figure 2 - Exonuclease footprinting analysis revealing drug-specific differences in platination site selectivity between naked and nucleosomal DNA.

NCP and naked DNA were treated with cisPt or oxPt (color-coded), followed by end labeling of the purified DNA and exonuclease III digestion (Supplementary Methods). Before fragment separation by denaturing gel electrophoresis, samples were deplatinated with thiourea to eliminate migration retardation resulting from the presence of adducts⁸. This allows determination of adduct sites at approximately base-pair resolution in comparison with a modified Maxam-Gilbert purine-sequencing standard (m), in which the 3'-phosphate groups have been removed by polynucleotide kinase treatment to yield the same 3'-OH ends that arise from exonuclease cleavage. Red arrow denotes central bases. (a–c) Denaturing PAGE of exonuclease-treated DNA samples shows digest termination sites resulting from encounter of platinum adducts. Overall footprint (a) and resolved sections corresponding to the central (b) and 3' (c) regions are shown. (d) DNA sequence for 79 of 147 base pairs. Regions where the DNA minor groove faces inward, toward the histone octamer, are colored orange. Exonuclease stop sites are depicted as arrowheads adjacent to the terminal 3' nucleotide, pointing toward the apparent platinum adduct. Filled symbols indicate relatively strong termination points, and open symbols indicate moderate termination points.

*Note: In the version of this article initially published online, dash marks indicating the position of molecular weight markers in Figure 2a are missing. The error has been corrected for all versions of the article.