Kumari Anuradha, Minko Irina G, Harbut Michael B, Finkel Steven E, Goodman Myron F, Lloyd R Stephen
Center for Research on Occupational and Environmental Toxicology and the Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon 97239-3098.
Molecular and Computational Biology Program, Department of Biological Sciences, Los Angeles, California 90089-2910.
J Biol Chem. 2008 Oct 10;283(41):27433-27437. doi: 10.1074/jbc.M801237200. Epub 2008 Aug 11.
Repair of interstrand DNA cross-links (ICLs) in Escherichia coli can occur through a combination of nucleotide excision repair (NER) and homologous recombination. However, an alternative mechanism has been proposed in which repair is initiated by NER followed by translesion DNA synthesis (TLS) and completed through another round of NER. Using site-specifically modified oligodeoxynucleotides that serve as a model for potential repair intermediates following incision by E. coli NER proteins, the ability of E. coli DNA polymerases (pol) II and IV to catalyze TLS past N(2)-N(2)-guanine ICLs was determined. No biochemical evidence was found suggesting that pol II could bypass these lesions. In contrast, pol IV could catalyze TLS when the nucleotides that are 5' to the cross-link were removed. The efficiency of TLS was further increased when the nucleotides 3' to the cross-linked site were also removed. The correct nucleotide, C, was preferentially incorporated opposite the lesion. When E. coli cells were transformed with a vector carrying a site-specific N(2)-N(2)-guanine ICL, the transformation efficiency of a pol II-deficient strain was indistinguishable from that of the wild type. However, the ability to replicate the modified vector DNA was nearly abolished in a pol IV-deficient strain. These data strongly suggest that pol IV is responsible for TLS past N(2)-N(2)-guanine ICLs.
大肠杆菌中链间DNA交联(ICL)的修复可通过核苷酸切除修复(NER)和同源重组相结合的方式进行。然而,有人提出了另一种机制,即修复由NER启动,随后进行跨损伤DNA合成(TLS),并通过另一轮NER完成。使用位点特异性修饰的寡脱氧核苷酸作为大肠杆菌NER蛋白切割后潜在修复中间体的模型,测定了大肠杆菌DNA聚合酶(pol)II和IV催化TLS越过N(2)-N(2)-鸟嘌呤ICL的能力。未发现生化证据表明pol II可以绕过这些损伤。相反,当交联位点5'端的核苷酸被去除时,pol IV可以催化TLS。当交联位点3'端的核苷酸也被去除时,TLS的效率进一步提高。正确的核苷酸C优先掺入损伤对面。当用携带位点特异性N(2)-N(2)-鸟嘌呤ICL的载体转化大肠杆菌细胞时,pol II缺陷菌株的转化效率与野生型菌株无异。然而,在pol IV缺陷菌株中,复制修饰载体DNA的能力几乎丧失。这些数据强烈表明,pol IV负责越过N(2)-N(2)-鸟嘌呤ICL的TLS。