Methylmethanesulfonate-induced DNA damage and its repair in cultured human fibroblasts: normal rates of induction and removal of alkali-labile sites in xeroderma pigmentosum (group A) cells

Abstract

Using conventional alkaline sucrose sedimentation analysis, we have compared the initial yield and subsequent enzymatic repair of DNA damage induced in cultured human [normal (GM38 and GM43) and xeroderma pigmentosum (XP12BE)] fibroblasts by the monofunctional alkylating agent methylmethanesulfonate (MMS). Exposure of both cell types to MMS (0-20 mM) resulted in a linear dose-response relationship for the formation of DNA alkali-labile sites (i.e. structurally altered sites that appeared as single-strand interruptions at alkaline pH). The majority (approximately 90%) of the sites detected in the normal cells immediately after chemical treatment (less than or equal to 8 mM) disappeared rapidly, with a half-life of less than or equal to 3 h; the remainder, however, persisted in genomic DNA for at least 72 h. Approximately 40% of the alkali-labile sites induced by 5 mM MMS could be stabilized by methoxyamine, a chemical which is known to react with apurinic/apyrimidinic (AP) sites in DNA so as to prevent alkali-catalyzed beta-elimination; thus this fraction of the alkali-labile sites, which is estimated to constitute approximately 4% of the total genomic injury inflicted by the chemical, may be ascribable to AP sites. XP12BE cells responded normally to MMS exposure as judged by: (i) the rate of initial induction of alkali-labile sites, including those (AP sites) subject to methoxyamine stabilization; (ii) the incidence of alkali-labile sites in cellular DNA at various times (0-72 h) after administration of the alkylating agent; and (iii) the capacity to execute the long-patch mode of excision repair as measured by accumulation of 1-beta-D-arabinofuranosylcytosine-induced strand breaks during post-treatment cell incubation. In addition, we have found that a significant portion of the genetic material in human fibroblasts undergoes degradation upon sustaining MMS damage, as indicated by the appearance of small DNA fragments (sedimenting near the top of alkaline sucrose gradients) in chemically treated cultures incubated for 24 h. Interestingly, the extent of this type of DNA injury proved to be markedly greater in XP12BE than in GM38 cells, and in exponentially growing than in G2-arrested normal cultures.