Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205, USA.
Biochemistry. 2010 May 11;49(18):3977-88. doi: 10.1021/bi902169q.
DNA interstrand cross-links (ICLs) are cytotoxic products of common anticancer drugs and cellular metabolic processes, whose mechanism(s) of repair remains poorly understood. In this study, we show that cross-link structure affects ICL repair in nonreplicating reporter plasmids that contain a mispaired N(4)C-ethyl-N(4)C (C-C), N3T-ethyl-N3T (T-T), or N1I-ethyl-N3T (I-T) ICL. The T-T and I-T cross-links obstruct the hydrogen bond face of the base and mimic the N1G-ethyl-N3C ICL created by bis-chloroethylnitrosourea, whereas the C-C cross-link does not interfere with base pair formation. Host-cell reactivation (HCR) assays in human and hamster cells showed that repair of these ICLs primarily involves the transcription-coupled nucleotide excision repair (TC-NER) pathway. Repair of the C-C ICL was 5-fold more efficient than repair of the T-T or I-T ICLs, suggesting the latter cross-links hinder lesion bypass following initial ICL unhooking. The level of luciferase expression from plasmids containing a C-C cross-link remnant on either the transcribed or nontranscribed strand increased in NER-deficient cells, indicating NER involvement occurs at a step prior to remnant removal, whereas expression from similar T-T remnant plasmids was inhibited in NER-deficient cells, demonstrating NER is required for remnant removal. Sequence analysis of repaired plasmids showed a high proportion of C residues inserted at the site of the T-T and I-T cross-links, and HCR assays showed that Rev1 was likely responsible for these insertions. In contrast, both C and G residues were inserted at the C-C cross-link site, and Rev1 was not required for repair, suggesting replicative or other translesion polymerases can bypass the C-C remnant.
DNA 链间交联（ICLs）是常见抗癌药物和细胞代谢过程中的细胞毒性产物，其修复机制仍知之甚少。在这项研究中，我们表明交联结构会影响非复制报告质粒中 ICL 的修复，这些质粒包含错配的 N(4)C-乙基-N(4)C（C-C）、N3T-乙基-N3T（T-T）或 N1I-乙基-N3T（I-T）ICL。T-T 和 I-T 交联阻碍了碱基的氢键面，模拟了双氯乙基亚硝脲产生的 N1G-乙基-N3C ICL，而 C-C 交联不干扰碱基对形成。人源和仓鼠细胞的宿主细胞再激活（HCR）测定表明，这些 ICL 的修复主要涉及转录偶联核苷酸切除修复（TC-NER）途径。C-C ICL 的修复效率比 T-T 或 I-T ICL 高 5 倍，表明后两种交联阻碍了初始 ICL 解钩后的损伤绕过。在 NER 缺陷细胞中，含有转录或非转录链上 C-C 交联残余物的质粒中的荧光素酶表达水平增加，表明 NER 参与发生在残余物去除之前的步骤，而类似 T-T 残余物质粒的表达在 NER 缺陷细胞中受到抑制，表明 NER 是残余物去除所必需的。修复质粒的序列分析显示，T-T 和 I-T 交联处插入了大量 C 残基，HCR 测定表明 Rev1 可能负责这些插入。相比之下，C-C 交联处既插入了 C 残基，也插入了 G 残基，而 Rev1 修复不需要，这表明复制或其他跨损伤聚合酶可以绕过 C-C 残余物。
