Fujiwara Y
Cancer Res. 1975 Oct;35(10):2780-9.
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.
对来自小鼠L细胞的DNA进行的掺入和碱性蔗糖沉降研究已经证明了N-甲基-N-亚硝基脲（MNU）和甲磺酸甲酯（MMS）的以下作用。增加这两种试剂的浓度会增加现有DNA的单链断裂或碱不稳定损伤的数量，这通过降低其相对速率来影响[3H]胸苷掺入DNA。在MNU处理后于[3H]胸苷中第1小时新合成的DNA，其分子量低于处理细胞中具有碱不稳定损伤的现有DNA，也低于对照细胞中合成的DNA。在处理细胞中形成的这种小片段在随后用胸苷或5-溴-2'-脱氧尿苷进行的4小时追踪中被延长并连接形成高分子量DNA。近紫外光解选择性地将5-溴-2'-脱氧尿苷延长的DNA降解为几乎与追踪前一样小的片段。此外，在胸苷追踪期间存在的咖啡因（2 mM）会阻止新生链的延长，尽管在MNU烷基化细胞中部分发生了对咖啡因不敏感的链生长。现有DNA中的MMS损伤（碱中的单链断裂）也会暂时中断复制合成并产生短片段，但其延长似乎对咖啡因不敏感。我们的结果表明，MNU可能像紫外线损伤一样产生对咖啡因敏感的中断（可能是缺口）以及新生链中对咖啡因不敏感的脱嘌呤位点定向中断，而MMS可能仅导致后者。对此的进一步证据是，与紫外线杀伤一样，咖啡因仅增强MNU对L细胞的杀伤作用。这种特定MNU损伤的程度估计不超过甲基化总程度的4%，预测对咖啡因敏感修复可及的损伤将是除N7-甲基鸟嘌呤之外的次要产物。MNU的诱变和致癌作用高于MMS，这可能归因于这种特定的MNU损伤及其修复。
