Extracts of xeroderma pigmentosum group a fibroblasts introduce less nicks into methyl methanesulfonate-treated dna than extracts of normal fibroblasts

Abstract

Summary Endonuclease activities with methyl methanesulfonate (MeSO 2 OMe)-treated and depurinated double-stranded DNA as substrates were determined in cell-free extracts of 3 Xeroderma pigmentosum (XP) group A fibroblast cell lines and 3 normal ones. Approximately equal levels of endonuclease were found using depurinated DNA, the apparent K m being 8.3 apurinic sites per PM2 molecule. V max was 2.1 breaks/PM2 molecule/min/ μ g protein. The use of MeSO 2 OMe-treated DNA as substrate, however, revealed that the XP group A extracts introduced less than 40% of the single-strand breaks obtained with extracts of normal cells. Relating this endonucleolytic cleavage to the number of alkali-labile (i.e., apurinic) sites present in MeSO 2 OMe-treated DNA, an apparent K m of 50 apurinic sites per PM2 molecule for both normal and XP extracts was calculated; V max values were 4.6 and 1.7 breaks/PM2 molecule/min/ μ g protein, respectively. These results suggest 2 possible differences between normal and XP group A fibroblasts: (a) XP group A cells contain lower amounts of such enzyme(s) as DNA glycosylase which acts directly on methylated DNA, or (b) the enzyme(s) cleaving methylated DNA are altered in XP group A cells in such a fashion as to make them more amenable to inhibition under our assay conditions.