Sidik K, Smerdon M J
Carcinogenesis. 1984 Feb;5(2):245-53. doi: 10.1093/carcin/5.2.245.
We have examined both the initial nuclease sensitivity and subsequent nucleosome rearrangement of newly repaired regions of chromatin in human diploid fibroblasts treated with methyl methanesulfonate (MMS) and methylnitrosourea (MNU). We initially examined the effect of these two alkylating agents on DNA replicative synthesis. The results indicate that immediately following damage by MMS or MNU, at a concentration of 2 mM, the level of replicative synthesis is 20-25% of the level in untreated cells. In the MMS-treated cells, this suppression of replicative synthesis is short lived and by 15 h after damage the level of replicative synthesis is approximately 3-fold greater than that in untreated cells. This 'latent stimulation' of replicative synthesis was not observed in the cells treated with 2 mM MNU, although the level of replicative synthesis in these cells did approach the level of untreated cells at later times. When these contributions were corrected for, it was found that the nucleotides incorporated by repair synthesis are initially (i.e., immediately following repair synthesis) both staphylococcal nuclease and DNase I sensitive, and are underrepresented in isolated nucleosome core DNA. Using methods previously described by us, we show that the relative nuclease sensitivity of these regions is quantitatively similar to that of newly repaired DNA following damage by u.v. radiation. Furthermore, the relative nuclease sensitivity of newly repaired DNA is initially high regardless of the time after damage that repair occurs (at least for 13 h after damage). This feature is also similar to u.v. induced repair synthesis. Finally, pulse-chase experiments demonstrated that following repair synthesis induced by MMS or MNU rearrangements of chromatin structure take place, and both the rate and extent of these rearrangements are similar to that observed for cells treated with u.v. radiation or bulky chemical carcinogens. Thus, our results indicate that the excision repair induced by these two small alkylating agents is associated with the same overall chromatin structural features as the excision repair of DNA damage induced by u.v. radiation and 'u.v.-mimetic' chemicals.
我们研究了用甲磺酸甲酯（MMS）和甲基亚硝基脲（MNU）处理的人二倍体成纤维细胞中染色质新修复区域的初始核酸酶敏感性和随后的核小体重排。我们首先研究了这两种烷化剂对DNA复制合成的影响。结果表明，在2 mM浓度下，MMS或MNU损伤后立即进行的复制合成水平是未处理细胞中水平的20 - 25%。在MMS处理的细胞中，这种复制合成的抑制是短暂的，损伤后15小时，复制合成水平比未处理细胞高约3倍。在用2 mM MNU处理的细胞中未观察到这种复制合成的“潜在刺激”，尽管这些细胞中的复制合成水平在后期确实接近未处理细胞的水平。当对这些因素进行校正后发现，修复合成掺入的核苷酸最初（即修复合成后立即）对葡萄球菌核酸酶和DNase I敏感，并且在分离的核小体核心DNA中含量不足。使用我们之前描述的方法，我们表明这些区域的相对核酸酶敏感性在数量上与紫外线辐射损伤后新修复的DNA相似。此外，无论修复发生在损伤后的时间如何（至少在损伤后13小时内），新修复DNA的相对核酸酶敏感性最初都很高。这一特征也与紫外线诱导的修复合成相似。最后，脉冲追踪实验表明，MMS或MNU诱导的修复合成后会发生染色质结构重排，这些重排的速率和程度与用紫外线辐射或大分子化学致癌物处理的细胞中观察到的相似。因此，我们的结果表明，这两种小烷化剂诱导的切除修复与紫外线辐射和“紫外线模拟”化学物质诱导的DNA损伤切除修复具有相同的整体染色质结构特征。
