1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980-995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.

中文翻译：

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在顺铂存在下，DNA修复蛋白O6-烷基鸟嘌呤DNA烷基转移酶与DNA的交联。

1,1,2,2-顺二氨二氯铂（II）（顺铂）是一种化学治疗剂，在临床上广泛用于治疗各种癌症。顺铂的抗肿瘤活性通常归因于其通过在DNA双链体中两个亲核位点的顺序电镀形成链内和链间DNA-DNA交联的能力。但是，顺铂还诱导DNA蛋白质损伤（DPC），由于其阻断DNA复制和转录的能力，可能会对其生物学效应有所贡献。我们之前曾报道过250多个核蛋白，包括高迁移率基团蛋白，组蛋白和延伸因子在顺铂处理的人HT1080细胞中形成了DPC（Ming等人.Chem.Res.Toxicol.2017，30，980-995）。有趣的是，加热后逆转了顺铂诱导的DNA-蛋白质偶联物，通过未知的机制。在目前的工作中，DNA修复蛋白O6-烷基鸟嘌呤DNA烷基转移酶（AGT）被用作研究顺铂介导的DNA-蛋白交联的分子细节并建立其逆转机理的模型。我们发现，在存在顺铂的情况下，AGT很容易与DNA交联。源自dG-Pt-AGT复合物的胰蛋白酶肽的HPLC-ESI + -MS / MS测序表明，该交联发生在该蛋白内的六个位点，包括Glu110，Lys125，Cys145，His146，Arg147和Cys150。顺铂诱导的Lys-Gua交联（1,1-顺二氨-2-（5-氨基-5-羧基戊基）氨基-2-（2'-脱氧鸟苷-7-基）-铂（II）（dG与相应的真实标准品相比，通过HPLC-ESI + -MS / MS检测修饰蛋白的总消化物（-Pt-Lys）。dG-Pt-AGT复合物经历了从蛋白质到DNA的电镀迁移，形成了顺式[[Pt（NH3）2 {d（GpG）}]交联，可通过HPLC-ESI + -MS / MS检测到。我们的结果为顺铂介导的DNA-蛋白质交联的机理及其与相应DNA-DNA损伤的动态平衡提供了新的见解。