Decreased cisplatin damage-dependent DNA synthesis in cellular extracts ofmismatch repair deficient cells

The proficiency of both nucleotide excision repair (NER) and DNA mismatch r epair (MMR) influences cellular sensitivity to cisplatin (cis-diamminedichl oroplatinum). To gain further insight into how MMR may influence platinum d rug sensitivity, the effect of loss of MMR on repair synthesis was measured in vitro by a commonly used method that relies on whole-cell extracts to d rive [alpha-P-32]dATP incorporation into cisplatin-damaged plasmid DNA. Ext racts evaluated include those from cells with or without functional hMLH1 ( HCT116+ch2 versus HCT116+ch3, respectively) and hMSH2 (HEC59 versus HEC59+c h2, respectively). Loss of MMR in the HCT116 system was associated with a 2 .8-fold reduction in cisplatin damage-specific DNA synthesis, whereas it wa s associated with a 3.0-fold reduction in the HEC59 system, suggesting that a decrease in the ability to repair cisplatin damaged DNA accompanies loss of MMR. An in vitro DNA excision assay that utilized a substrate containin g a site-specific cisplatin adduct was performed. Using this highly NER-spe cific assay, no significant difference was apparent between the extracts de rived from NER-proficient versus-deficient cells. These and other data lead us to suggest that the increase in apparent repair synthesis in platinum-d amaged plasmids by extracts from MMR-proficient versus-deficient cellular e xtracts may reflect a distinct and possibly adverse DNA synthetic process r ather than productive NER. BIOCHEM PHARMACOL 57;8:861-867, 1999. (C) 1999 E lsevier Science Inc.