位点特异性o6 -烷基鸟嘌呤dna -烷基转移酶-寡核苷酸交联的合成与表征

Pratibha P. Ghodke, Matthew E. Albertolle, Kevin M. Johnson, F. Peter Guengerich
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引用次数: 5

摘要

o6 -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在大肠杆菌和哺乳动物细胞中异种表达时,AGT- dna交联具有诱变性。为了了解生物学后果,需要可靠地获得agt -寡核苷酸交联。本文介绍了脱氧鸟苷n2位和脱氧腺苷n6位特异性agt寡核苷酸交联的合成和表征。我们开发了一种合成丙炔修饰寡核苷酸的后寡聚化策略。以铜催化叠氮化物-炔环加成为关键步骤,得到了与AGT活性位点半胱氨酸-145交联反应的良好亲电试剂碘乙酰胺连接寡核苷酸。AGT胰蛋白酶化和寡核苷酸水解,结合液相色谱-串联质谱分析,确定了核碱基内合肽。该方法为位点特异性DNA-蛋白交联的合成和表征提供了一种有用的策略,可进一步用于了解蛋白水解降解耦合DNA修复机制。©2019 by John Wiley &儿子,Inc。
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Synthesis and Characterization of Site-Specific O6-Alkylguanine DNA-Alkyl Transferase-Oligonucleotide Crosslinks

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.

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Current Protocols in Nucleic Acid Chemistry
Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry
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期刊介绍: Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.
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