Postreplication repair of alkylation damage to DNA of mammalian cells in culture

Abstract

Incorporation and alkaline sucrose sedimentation studies of DNA from mouse L-cells have demonstrated the following effects of N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS). Increasing the concentration of both agents increases the number of single-strand breaks or alkali-labile lesions of existing DNA, which affects the incorporation of [3H]thymidine into DNA by reducing its relative rate. DNA that is newly synthesized during the 1st hr in [3H]thymidine after MNU treatment is of lower molecular weight than is existing DNA with alkali-labile lesions in treated cells and is also lower than DNA synthesized in control cells. Such small segments formed in treated cells are elongated and joined to form high-molecular-weight DNA in the subsequent 4-hr chase in thymidine or 5-bromo-2'-deoxyuridine. Near-ultraviolet photolysis selectively degrades 5-bromo-2'-deoxyuridine-elongated DNA to segments that are nearly as small as those before chase. Further, caffeine (2 mM) present during the thymidine chase prevents nascent-strand elongation, although caffeine-insensitive chain growth occurs partly in MNU-alkylated cells. The MMS lesion (single-strand breakage in alkali) in existing DNA also temporarily interrupts replicative synthesis and makes short segments, but their elongation seems insensitive to caffeine. Our results indicate that MNU may produce both caffeine-sensitive interruptions (probably gaps), as ultraviolet damage does, and apurinic site-directed, caffeine-insensitive interruptions in nascent strands, while MMS may cause exclusively the latter. Further evidence for this is the caffeine potentiation of only MNU killing, like ultraviolet killing, of L-cells. The extent of such a specific MNU lesion is estimated to be no more than 4% of the total extent of methylation, predicting that the lesion that is accessible to caffeine-sensitive repair will be a minor product(s) other than N7-methylguanine. Mutagenic and carcinogenic effects of MNU, which are higher than those of MMS, could be ascribed to such a particular MNU lesion(s) and its repair.