哺乳动物细胞对丝裂霉素诱导的DNA链间交联的修复

目的:探讨哺乳动物细胞能否修复DNA链间交联(interstrand crosslink,ICL).方法:构建带有丝裂霉素(mitomycin C,MMC)交联的质粒pCMV-Luc/ICL,将该交联质粒转染无修复酶缺陷和有修复酶缺陷的哺乳动物细胞系,观察细胞对MMC链间交联的修复能力.结果:(1)无酶缺陷的哺乳动物细胞对DNA的修复能力很强,即使缺乏同源序列,也能有效地去除DNA链间交联;(2)修复过程需要许多酶类参与,其中与核苷酸切除修复(nucleotide excision repair,NER)相关的酶类起着关键性作用,提示NER参与MMC链间交联的修复过程;(3)序列分析表明,非同源性重组修复为易错修复(error-prone repair).结论:哺乳动物细胞能有效地修复MMC诱导的DNA链间交联,核苷酸切除修复是重要的修复途径.

Repair of Mitomycin-C Induced DNA Interstrand Cross-Link in Mammalian Cells

Objective: To study the repair mechanisms of ICLs in mammalian cells. Methods: A site-specific MMC cross-link was placed between the promoter and the coding region to inactivate the expression of luciferase genes from reporter plasmids. An in vivo reactivation assay was developed to examine the removal of ICLs in cultured cells. Results: MMC cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the NER pathway were examined and found to be highly defective in the recombination independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near the positions of MMC crosslinks. Conclusion: Recombination-independent ICL pathway exists in mammalian cells and NER involve in the repair with an error-prone mechanism.