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Figure 2.
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 adducts^8. 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.
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