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

Abstract

O 6 -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. C 2019 by John Wiley & Sons, Inc.Keywords: copper-catalyzed azide-alkyne cycloaddition r DNA-protein crosslinks r O 6 -alkylguanine DNA-alkyltransferase r oligonucleotides r postoligomerization r proteomics

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

“…The same scenario was observed previously in the analysis of the DNA-AGT cross-link. Proteomic analysis of AGT- N 6 -DNA cross-link indicated the presence of a singly oxidized sulfur during in-gel (6.5% of oxidized product) as well as in-solution (0.8% of oxidized product) digestion by trypsin (data not shown) ( 43 ). We had not observed the presence of sulfoxides or sulfones in our analysis of AGT tryptic peptides cross-linked to DNA in previous work ( 50 , 51 ), but these assays were done more rapidly than the synthetic work described here and we had set up our searches based only on the thioether product.…”

“…S8 ). The cross-link (100 pmol) was dried and suspended in HF (48%, 50 μl), and incubated at 4 °C for 14 h ( 43 ). The sample was dried under a stream of nitrogen, resuspended in anhydrous CH 3 OH (50 μl), and dried again under a stream of nitrogen.…”

“…O 6 -Alkylguanine DNA-alkyltransferase (AGT, or MGMT), a ∼22 kDa protein, is a crucial repair system for protection from alkylating agents ( 43 ). It directly removes and transfers alkyl groups from the O6 position on guanine to the Cys-145 located in its active site.…”

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides

“…2 ). The synthesis was carried out on a CPG (controlled pore glass) level, which involves nucleophilic displacement using cystamine followed by O 6 -( p -nitrophenylethyl) (NPE) deprotection ( 41 ). The final oligonucleotide deprotection was carried out to cleave CPG beads and to remove base-protecting groups to obtain N 2 -cystamine-dG-modified oligonucleotide ( Fig.…”

“…The dried pellet of hydrolyzed oligonucleotide-peptide cross-links was dissolved in 0.1% HCO2H (20 µl, v/v), shaken for 10 min, and centrifuged for 5 min at 23 °C (21,000  g). Finally, nucleobaseadducted peptides (without any purification) were analyzed using a nanoLC Ultra system (Eksigent Technologies, Dublin, CA) interfaced with an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) in the positive ion mode as described previously (41), except for the separation conditions: a linear gradient increased from 2% to 45% solvent B over a period of 0 to 45 min, increased from 45% to 95% solvent B over a period of 45 to 50 min, held at 95% solvent B over a period of 50 to 60 min, decrease from 95% to 2% solvent B over a period of 60 to 62 min and column was equilibrated at 2% solvent B over a period of 62 to 72 min (all v/v).…”