Laboratory of Molecular Biology and DNA repair, Department of Medicine (DAME), University of Udine, Udine, Italy.
Methods Mol Biol. 2023;2701:21-38. doi: 10.1007/978-1-0716-3373-1_2.
APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is a central enzyme of the base excision repair (BER) pathway playing a pivotal role in protecting mammalian cells against genotoxins and in safeguarding genome stability. Recently, we demonstrated the APE1 ability to process abasic ribonucleotides embedded in DNA. Here, we provide a pipeline of protocols to quantify endodeoxyribonuclease activity by APE1 on these substrates, by using recombinant protein and whole-cell extracts. The repair capacity is measured by using fluorescent oligonucleotide substrates, which are then separated by polyacrylamide gel electrophoresis and detected by imaging scanning. The specificity of APE1 action is demonstrated using specific APE1 enzymatic inhibitors.
APE1（脱嘌呤/脱嘧啶核酸内切酶 1）是碱基切除修复（BER）途径的核心酶，在保护哺乳动物细胞免受遗传毒素和维持基因组稳定性方面发挥着关键作用。最近，我们证明了 APE1 能够处理嵌入 DNA 中的碱基缺失核糖核苷酸。在这里，我们提供了一个使用重组蛋白和全细胞提取物来定量 APE1 对这些底物进行内切核酸酶活性的方案。通过使用荧光寡核苷酸底物来测量修复能力，然后通过聚丙烯酰胺凝胶电泳将其分离，并通过成像扫描进行检测。通过使用特定的 APE1 酶抑制剂来证明 APE1 作用的特异性。
