EVIDENCE FOR AP SITE FORMATION RELATED TO DNA-OXYGEN ALKYLATION IN CHO CELLS TREATED WITH ETHYLATING AGENTS
EVIDENCE FOR AP SITE FORMATION RELATED TO DNA-OXYGEN ALKYLATION IN CHO CELLS TREATED WITH ETHYLATING AGENTS
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DOI：
10.1016/0921-8777(90)90040-c
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发表时间：
1990-07-01
期刊：
影响因子：
--
通讯作者：
DOGLIOTTI, E
中科院分区：
文献类型：
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作者：
FORTINI, P;BIGNAMI, M;DOGLIOTTI, E
n -乙基-n -亚硝基脲（ENU）在CHO细胞中诱导的DNA单链断裂（ssb）在处理后10分钟内迅速重新封闭。这种快速修复动力学不能用碱基切除修复的速率来解释，碱基切除修复去除半衰期为几小时的主要乙基产物。我们已经探索了甲氧基胺（MX）的潜在用途，甲氧基胺是一种化学物质，在中性pH下与体外DNA中的AP位点发生反应，以澄清enu诱导的ssb的起源。在用硫酸二乙酯（DES）处理细胞时，50 mM MX的存在导致碱基切除修复过程中产生的AP位点的修复选择性抑制，并抑制了这些位点的碱性切割。在MX存在的情况下，用ENU处理CHO细胞清楚地表明，处理后立即观察到的ssb爆发是由于AP位点的形成。体外ENU处理的质粒DNA不存在AP内切酶敏感位点；因此，ENU处理在CHO细胞中产生的AP位点不是由于一种非常不稳定的乙基加合物的化学水解，而是一种尚未定义的酶促途径的中间体。这一过程在sn1型乙基化剂（ENU和n -乙基-n " -硝基-n -亚硝基胍）处理后特别发生，表明这一现象与dna -氧烷基化有关。我们认为这些断裂是由o6 -乙基鸟嘌呤加工机制产生的，而不去除修饰的碱基。
DNA single-strand breaks (ssb) induced by N-ethyl-N-nitrosourea (ENU) in CHO cells are quickly resealed within 10 min after treatment. This rapid repair kinetics is not explained by the rate of base excision repair which removes the main ethyl products with a half-life in the order of hours. We have explored the potential use of methoxyamine (MX), a chemical that reacts at neutral pH with AP sites in DNA in vitro, to clarify the origin of ENU-induced ssb. The presence of 50 mM MX during cell treatment with diethyl sulfate (DES) caused selective inhibition of the repair of AP sites generated during base excision repair and inhibited alkaline cleavage at these sites. The treatment of CHO cells with ENU in the presence of MX clearly showed that the burst of ssb observed immediately after treatment was due to AP site formation. Plasmid DNA treated in vitro with ENU did not present AP endonuclease-sensitive sites; therefore, the AP sites produced in CHO cells by ENU treatment are not due to the chemical hydrolysis of a very unstable ethyl adduct but rather are intermediates of an as yet undefined enzymatic pathway. This process occurs specifically after treatment with SN1-type ethylating agents (ENU and N-ethyl-N''-nitro-N-nitrosoguanidine) suggesting an association between this phenomenon and DNA-oxygen alkylation. We suggest that these breaks are generated by a mechanism of O6-ethylguanine processing without removal of the modified base.