Ghodke Pratibha P, Albertolle Matthew E, Johnson Kevin M, Guengerich F Peter
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee.
Curr Protoc Nucleic Acid Chem. 2019 Mar;76(1):e74. doi: 10.1002/cpnc.74. Epub 2019 Jan 18.
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.
O-烷基鸟嘌呤DNA烷基转移酶（AGT）是一种DNA修复蛋白，可与DNA形成交联。已知当AGT在大肠杆菌以及哺乳动物细胞中异源表达时，AGT-DNA交联具有致突变性。为了解其生物学后果，需要可靠地获得AGT-寡核苷酸交联物。本文描述了在脱氧鸟苷的N2位和脱氧腺苷的N6位上位点特异性AGT-寡核苷酸交联物的合成与表征。我们开发了一种用于合成炔丙基修饰寡核苷酸的寡聚后策略。铜催化的叠氮化物-炔烃环加成反应被用作关键步骤，以获得碘乙酰胺连接的寡核苷酸，这些寡核苷酸可作为与AGT活性位点的半胱氨酸-145进行交联反应的良好亲电试剂。对AGT进行胰蛋白酶消化和对寡核苷酸进行水解，并结合液相色谱-串联质谱分析，用于确认碱基加合的肽段。该方法为位点特异性DNA-蛋白质交联物的合成与表征提供了一种有用的策略，可进一步用于理解蛋白水解降解偶联的DNA修复机制。©2019约翰威立国际出版公司。
