Hjertvik M, Erixon K, Ahnström G
Department of Radiobiology, Stockholm University, Sweden.
Mutat Res. 1998 Mar;407(2):87-96. doi: 10.1016/s0921-8777(97)00062-1.
Alkylating agents have been reported to give rise to both short and long patches of repair. The reason for the different patch sizes is not known. One possibility is that alkylating agents can trigger both base and nucleotide excision repair. Another possibility is that base excision repair itself can result in different patch sizes. Recognition and incision at lesions is the rate limiting step in excision repair. In order to discriminate between base and nucleotide excision repair it would be desirable to be able to distinguish between different incision activities. In order to accurately measure incision rates, the rejoining of the strand-breaks formed must be inhibited. We have used two inhibitors, aphidicolin and 3-aminobenzamide. Aphidicolin, an inhibitor of DNA polymerases alpha/delta/epsilon. caused accumulation of single-strand breaks both after UV and dimethylsulphate. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose)-polymerase caused accumulation of single-strand breaks only after alkylating agents and is thus specific for base excision repair. Enzymatic activities can be characterised by their activation energy. In order to discriminate between base and nucleotide excision repair the temperature dependence of incision activities was determined. When the temperature is decreased, the incision rate is reduced to a larger extent for UV than for DMS-induced repair. Incisions in UV-irradiated cells are practically cut off at temperatures of 15 degrees C and below, whereas DMS-exposed cells still are actively repairing at this temperature. In DMS treated cells the temperature dependence was the same whether aphidicolin or 3-aminobenzamide was used, speaking against an involvement of nucleotide excision repair. In addition, cell lines deficient in nucleotide excision repair responded in the same way to aphidicolin after DMS treatment as normal cells and were able to make incisions at 15 degrees C. This indicates that nucleotide excision repair is not to any significant amount involved in repair of DNA damage induced by a methylating agent.
据报道，烷化剂可引发短片段和长片段修复。修复片段大小不同的原因尚不清楚。一种可能性是烷化剂可同时触发碱基切除修复和核苷酸切除修复。另一种可能性是碱基切除修复本身可导致不同大小的片段。损伤的识别和切割是切除修复中的限速步骤。为了区分碱基切除修复和核苷酸切除修复，能够区分不同的切割活性将是很有必要的。为了准确测量切割速率，必须抑制所形成的链断裂的重新连接。我们使用了两种抑制剂，阿非迪霉素和3-氨基苯甲酰胺。阿非迪霉素是DNA聚合酶α/δ/ε的抑制剂，在紫外线和硫酸二甲酯处理后均导致单链断裂的积累。3-氨基苯甲酰胺是聚（ADP-核糖）聚合酶的抑制剂，仅在烷化剂处理后导致单链断裂的积累，因此对碱基切除修复具有特异性。酶活性可通过其活化能来表征。为了区分碱基切除修复和核苷酸切除修复，测定了切割活性的温度依赖性。当温度降低时，紫外线诱导的修复中切割速率的降低程度比硫酸二甲酯诱导的修复更大。紫外线照射细胞中的切割在15摄氏度及以下的温度下几乎停止，而硫酸二甲酯处理的细胞在该温度下仍在积极修复。在硫酸二甲酯处理的细胞中，无论使用阿非迪霉素还是3-氨基苯甲酰胺，温度依赖性都是相同的，这表明核苷酸切除修复未参与其中。此外，核苷酸切除修复缺陷的细胞系在硫酸二甲酯处理后对阿非迪霉素的反应与正常细胞相同，并且能够在15摄氏度下进行切割。这表明核苷酸切除修复在很大程度上不参与甲基化剂诱导的DNA损伤的修复。
