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

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.

Keywords: AGT; Cisplatin; DNA-protein cross-links; Mass spectrometry.

Figure 1.

Detection of cisplatin-induced DNA-protein cross-links by gel electrophoresis. 12% SDS-PAGE analysis of ³²P-endlabeled DNA duplexes (5'-GGA GCT GGT GGC GTA GGC-3’ +strand) following incubation with (A) recombinant human AGT or (B) recombinant GAPDH protein in the presence of 1 (lane 4), 5 (lane 5), 10 (lane 6), 50 (lane 7) or 100 (lane 8) molar equivalents of cisplatin. Free duplex DNA (labeled “Free oligo”) migrates to the bottom of the gel, whereas DNA-protein cross-links display substantially reduced mobility.

Figure 2.

HPLC separation of reaction mixtures, following incubation of recombinant AGT protein with dG-Pt-Cl, as models for monoalkylated DNA to induce cross-linking. AGT-dG conjugates were identified by HPLC-ESI⁺-MS as shown in Figure 3.

Figure 3.

HPLC-ESI⁺-MS and deconvoluted spectra (inset) of (A) unreacted AGT protein, (B) dG-Pt-Cl monoadduct-treated AGT protein that had been separated by HPLC with retention time 32.1 min in Figure 2, and (C) dG-Pt-Cl monoadduct-treated AGT protein that had been separated by HPLC with retention time 30.1 min in Figure 2. Unmodified AGT (calculated, M = 21876 Da; observed, M = 21878 Da), AGT containing a single platinum cross-link to dG (calculated, M = 22354 Da; observed, M = 22355 Da or calculated, M = 22371 Da; observed, M = 22372 Da), AGT containing two platinum cross-links to dG (calculated, M = 22832 Da; observed, M = 22832 Da), and AGT containing three platinum cross-links to dG (calculated, M = 23327 Da; observed, M = 23328 Da).

Figure 4.

HPLC-ESI⁺-MS/MS analysis of tryptic peptides derived from cisplatin-induced AGT-dG conjugates at Glu¹¹⁰, Lys¹²⁵, Cys¹⁴⁵, His¹⁴⁶, Arg¹⁴⁷, and Cys¹⁵⁰. Fragment ions containing a cross-linked platinum adduct are indicated by “*”.

Figure 5

Crystal structure of human AGT protein bound to DNA (PDB 1T39) showing the sites of cisplatin-mediated DNA-protein cross-linking.

Figure 6.

Platination migration from AGT to dG. (A) Deconvoluted ESI⁺-MS spectra of dG-Pt-AGT that was incubated with dG for 10 min at 70 °C. (B) Spectra of dG-Pt-AGT that was incubated with dG for 60 min at 70 °C. (C) Spectra of dG-Pt-AGT complexes was incubated in the absence of dG for 10 min at 70 °C.

Figure 7.

HPLC-ESI⁺-MS/MS analysis of dG-Pt-dG conjugates produced as a result of platination migration from dG-Pt-AGT cross-links to dG. Extracted ion chromatogram of dG-Pt-dG (m/z 763.2 [M]⁺). Inset: MS/MS fragmentation. (A) Synthetic dG-Pt-dG; (B) Sample treated with dG for 60 min at 70 °C

Figure 8.

HPLC-ESI⁺-MS/MS analysis of dG-Pt-Lys conjugates in total proteolytic digests of AGT treated with dG-Pt-Cl to generate cross-links. Following HPLC purification of dG-Pt-AGT complexes, they were subjected to enzymatic digestion to release amino acid-nucleobase conjugates. (A) Synthetic dG-Pt-Lys; (B) Enzymatic digests of dG-Pt-AGT complexes.

Scheme 1.

Formation of bifunctional DNA adducts by cisplatin.

Scheme 2.

Mass spectrometry-based approach employed to characterize AGT-DNA cross-links of cisplatin and platination migration from AGT protein to dG to release intact AGT and form G-G cisplatin cross-links.