Title: Synthesis and Characterization of Site-Specific O6 -Alkylguanine DNA-Alkyl Transferase-Oligonucleotide Crosslinks.
Authors: Ghodke, Pratibha P; Albertolle, Matthew E; Johnson, Kevin M; Guengerich, F Peter
Published In Curr Protoc Nucleic Acid Chem, (2019 03)
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.
PubMed ID: 30657645
MeSH Terms: Catalysis; Catalytic Domain; Chromatography, Liquid/methods; Copper/chemistry; Cross-Linking Reagents/chemistry; Escherichia coli/genetics; O(6)-Methylguanine-DNA Methyltransferase/chemical synthesis*; O(6)-Methylguanine-DNA Methyltransferase/chemistry; O(6)-Methylguanine-DNA Methyltransferase/genetics; Oligonucleotides/chemistry*; Polymerization; Tandem Mass Spectrometry/methods; Templates, Genetic; Trypsin/chemistry