Synthesis and Characterization of Site-Specific O6 -Alkylguanine DNA-Alkyl Transferase-Oligonucleotide Crosslinks

Abstract

O⁶ -Alkylguanine DNA-alkyltransferase (AGT), a DNA repair protein, can form crosslinks with DNA. The AGT-DNA crosslinks are known to be mutagenic when AGT is heterologously expressed in Escherichia coli, as well as in mammalian cells. To understand the biological consequences, reliable access to AGT-oligonucleotide crosslinks is needed. This article describes the synthesis and characterization of site-specific AGT-oligonucleotide crosslinks at the N2-position of deoxyguanosine and N6-position of deoxyadenosine. We developed a post-oligomerization strategy for the synthesis of propargyl-modified oligonucleotides. Copper-catalyzed azide-alkyne cycloaddition was used as a key step to obtain the iodoacetamide-linked oligonucleotides, which serve as good electrophiles for the crosslinking reaction with cysteine-145 of the active site of AGT. Trypsinization of AGT and hydrolysis of oligonucleotides, combined with analysis by liquid chromatography-tandem mass spectrometry, was utilized to confirm the nucleobase-adducted peptides. This method provides a useful strategy for the synthesis and characterization of site-specific DNA-protein crosslinks, which can be further used to understand proteolytic degradation-coupled DNA repair mechanisms. © 2019 by John Wiley & Sons, Inc.

Keywords: DNA-protein crosslinks; O6-alkylguanine DNA-alkyltransferase; copper-catalyzed azide-alkyne cycloaddition; oligonucleotides; post-oligomerization; proteomics.

Figure 1.

Mechanism of DBE-derived AGT-DNA crosslink formation.

Figure 2.

Synthetic strategy of N²-dG oligonucleotide-AGT crosslink.

Figure 3.

Deconvoluted mass spectrum of N²-propargyl-dG modified oligonucleotide. Expected mass [M−H]⁻ 5791.812, Found [M−H]⁻ 5792.6001.

Figure 4.

(A) Sequence of the primer (FAM-labeled, 12-mer) and the template (19-mer); (B) SDS-PAGE (10%, Bis-Tris) of AGT-oligonucleotide crosslink reaction at the N2-position of dG. Lane 1: Precision Plus Protein dual color standard; Lane 2: AGT-oligonucleotide crosslink reaction after 1 h; Lane 3: AGT-oligonucleotide crosslink reaction after 3 h; Lane 4: AGT protein (21.9 KDa).

Figure 5.

Synthetic strategy of N⁶-dA oligonucleotide-AGT crosslink.

Figure 6.

Deconvoluted mass spectrum of N⁶-propargyl-dA-modified oligonucleotide. Expected mass [M−H]⁻ 5775.812, found [M−H]⁻ 5778.0005.

Figure 7.

(A) Sequence of the primer (12-mer) and the template (19-mer); (B) SDS-PAGE (10%, Bis-Tris) of AGT-cross-link reaction at the N6-position of dA. Lane 1: Protein Kaleidoscope standard; Lane 2: AGT-oligonucleotide crosslink reaction after 1 h; Lane 3: AGT-oligonucleotide crosslink reaction after 3 h; Lane 4: AGT protein (21.9 KDa).

Figure 8.

Flow chart of proteomics sample preparation: (A) for N2-dG oligonucleotide-AGT crosslink and (B) for N6-dA oligonucleotide-AGT crosslink.

Figure 9.

Peptide analysis of AGT-N2-dG oligonucleotide crosslink: (A) Observed structure of dodecamer guanine-adducted peptide (GNPVPILIPC*R) from the active site of AGT; (B) Annotated spectra of CID MS/MS of the [M+2H]2+ ion at m/z 822.9362 of the guanine-adducted peptide; (C) Extracted ion chromatogram of the guanine-adducted peptide for the [M+3H]3+ ion at m/z 548.9604. (C* = guanine-adducted Cys-145, observed an adduct with the guanine base only, due to the loss of deoxyribose).

Figure 10.

Peptide analysis of AGT N6-dA-oligonucleotide crosslink: (A) Observed structure of dodecamer adenine-adducted peptide (GNPVPILIPC*HR) from active site of AGT; (B) Annotated spectra of HCD MS/MS of the [M+2H]²⁺ ion at m/z 814.939 of adenine-adducted peptide; (C) Extracted ion chromatogram of adenine-adducted peptide for the [M+2H]³⁺ ion at m/z 543.627. (C* = adenine-adducted Cys-145, observed an adduct with the adenine base only, due to the loss of deoxyribose).

Figure 11.

Structures of known AGT-DNA crosslinks