Cross-linking of the DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of cisplatin

Xun Ming, Erin D. Michaelson-Richie, Arnold S. Groehler, Peter W. Villalta, Colin Campbell, Natalia Y. Tretyakova

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article number102840
JournalDNA Repair
Volume89
DOIs
StatePublished - May 2020

Bibliographical note

Funding Information:
We thank Professor Anthony E. Pegg (Penn State University) for generously providing AGT protein, Brock Matter (Masonic Cancer Center, University of Minnesota) for assistance with MS experiments, Jason A. Kuchar (Thermo Scientific) and Pratik Jagtap (Minnesota Supercomputing Insitute, University of Minnesota) for help with proteomic data analyses, Prof. Christine Chow and Bett Kimutai (Wayne State Univ.) for help with MS/MS data interpretation, and Robert Carlson for preparing figures for this manuscript. Funding for this research was provided by NIH grants R01-ES023350 and R01-CA-1006700 and a faculty development grant from the University of Minnesota Academic Health Center . X.M. and E.M.R. and were partially supported by the NIH Chemistry-Biology Interface Training Grant ( T32-GM08700 ), University of Minnesota Masonic Cancer Center , and University of Minnesota Graduate School . Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, funded in part by Cancer Center Support Grant CA-77598.

Funding Information:
We thank Professor Anthony E. Pegg (Penn State University) for generously providing AGT protein, Brock Matter (Masonic Cancer Center, University of Minnesota) for assistance with MS experiments, Jason A. Kuchar (Thermo Scientific) and Pratik Jagtap (Minnesota Supercomputing Insitute, University of Minnesota) for help with proteomic data analyses, Prof. Christine Chow and Bett Kimutai (Wayne State Univ.) for help with MS/MS data interpretation, and Robert Carlson for preparing figures for this manuscript. Funding for this research was provided by NIH grants R01-ES023350 and R01-CA-1006700 and a faculty development grant from the University of Minnesota Academic Health Center. X.M. and E.M.R. and were partially supported by the NIH Chemistry-Biology Interface Training Grant (T32-GM08700), University of Minnesota Masonic Cancer Center, and University of Minnesota Graduate School. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, funded in part by Cancer Center Support Grant CA-77598.

Publisher Copyright:
© 2020

Keywords

  • AGT
  • Cisplatin
  • DNA-protein cross-links
  • Mass spectrometry

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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