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

Ghodke PP, Albertolle ME, Johnson KM, Guengerich FP
Curr Protoc Nucleic Acid Chem. 2019 76 (1): e74

PMID: 30657645 · PMCID: PMC6504252 · DOI:10.1002/cpnc.74

O -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.

© 2019 John Wiley & Sons, Inc.

MeSH Terms (12)

Catalysis Catalytic Domain Chromatography, Liquid Copper Cross-Linking Reagents Escherichia coli O(6)-Methylguanine-DNA Methyltransferase Oligonucleotides Polymerization Tandem Mass Spectrometry Templates, Genetic Trypsin

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