Zhao Junhua, Jain Aklank, Iyer Ravi R, Modrich Paul L, Vasquez Karen M
Department of Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science-Park Research Division, Smithville, TX 78957, USA.
Nucleic Acids Res. 2009 Jul;37(13):4420-9. doi: 10.1093/nar/gkp399. Epub 2009 May 25.
DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA-RPA and XPC-RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSbeta were sensitive to psoralen ICLs. To further investigate the role of MutSbeta in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSbeta and NER proteins with Tdp-ICLs. We found that MutSbeta bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSbeta interacted with XPA-RPA or XPC-RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair.
DNA链间交联（ICL）是细胞毒性最强的DNA损伤类型之一，因此诸如补骨脂素等能诱导ICL的试剂是临床上有用的化疗药物。补骨脂素修饰的三链形成寡核苷酸（TFO）已被用于将ICL靶向特定基因组位点，以提高这些试剂的选择性。然而，TFO导向的补骨脂素ICL（Tdp-ICL）在人类细胞中是如何被识别和处理的尚不清楚。此前，我们报道了两种重要的核苷酸切除修复（NER）蛋白复合物，即XPA-RPA和XPC-RAD23B，在体外可识别ICL，并且DNA错配修复（MMR）复合物MutSβ缺陷的细胞对补骨脂素ICL敏感。为了进一步研究MutSβ在ICL修复中的作用以及MMR和NER途径的蛋白质在这些损伤上的潜在相互作用，我们用Tdp-ICL对MutSβ和NER蛋白进行了电泳迁移率变动分析和染色质免疫沉淀分析。我们发现，MutSβ在体外和体内均以高亲和力和特异性与Tdp-ICL结合，并且MutSβ在识别Tdp-ICL时与XPA-RPA或XPC-RAD23B相互作用。这些数据表明，MMR和NER途径的蛋白质在ICL的识别中相互作用，并提供了一个机制联系，通过该联系多个修复途径的蛋白质有助于ICL修复。
