Use of novel plasmid constructs to demonstrate fludarabine triphosphate inhibition of nucleotide excision repair of a site-specific 1,2-d(GpG) intrastrand cisplatin adduct.
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- Published online on: December 1, 1999 https://doi.org/10.3892/ijo.15.6.1177
- Pages: 1177-1260
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Abstract
We previously showed that fludarabine triphosphate (F-ara-ATP) acts as a potent inhibitor of nucleotide excision repair (NER). To determine how F-ara-ATP inhibits NER, we designed closed circular DNA duplexes, each containing a site-specific d(GpG) cisplatin adduct, as the substrate for an in vitro repair assay. We used the assay to determine the effects of F-ara-ATP on the incision, repair synthesis, and ligation steps in the NER process. A closed circular DNA duplex, pSSA, was first constructed by inserting an 87-bp oligonucleotide into pGEM-7Zf(+), from which a single-stranded plasmid (pTDS) was derived. The 87-bp insert included two potential repair patches; each contained a d(GpG) site flanked by unique sequences 22 nucleotides upstream and 6 nucleotides downstream so that four dAMP sites were strategically placed in patch 1 but were absent from patch 2. Each duplex substrate was then synthesized by annealing a unique primer containing a platinated and 32P-end-labeled oligonucleotide to the pTDS template, which was then converted to a duplex through polymerization and ligation. At 50 microM, F-ara-ATP inhibited repair synthesis; F-ara-ATP also inhibited incision and ligation, but only at concentrations of 200 microM and above. Chemical DNA sequencing of the repair patch revealed that F-ara-ATP induced the formation of a truncated repair patch in which DNA polymerization stopped one nucleotide before the first installed dAMP site - a potential site for F-ara-ATP's incorporation. In contrast, truncation of a repair patch was not detectable when the repair patch contained no dAMP. Taken together, the results suggest that F-ara-ATP induced patch truncation by self-incorporation and the incorporated F-ara-AMP was subsequently excised, presumably by polymerase-associated exonuclease activity. We conclude that F-ara-ATP blocks the NER process by strongly inhibiting DNA repair synthesis as well as by less strongly inhibiting incision and ligation. Our approach of designing plasmid constructs that contain sequence-specific repair patches with a strategically placed 32P label may provide a powerful tool for dissecting the mechanism of NER inhibition not only for F-ara-ATP but also for other NER inhibitors.
