In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site

Matilde Clarissa Malfatti; Giulia Antoniali; Gianluca Tell

doi:10.1007/978-1-0716-3373-1_2

In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site

Methods Mol Biol. 2023:2701:21-38. doi: 10.1007/978-1-0716-3373-1_2.

Authors

Matilde Clarissa Malfatti¹, Giulia Antoniali¹, Gianluca Tell²

Affiliations

¹ Laboratory of Molecular Biology and DNA repair, Department of Medicine (DAME), University of Udine, Udine, Italy.
² Laboratory of Molecular Biology and DNA repair, Department of Medicine (DAME), University of Udine, Udine, Italy. gianluca.tell@uniud.it.

PMID: 37574473
DOI: 10.1007/978-1-0716-3373-1_2

Abstract

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.

Keywords: Apurinic/apyrimidinic endodeoxyribonuclease 1; Base excision repair; Enzymatic activity; Inhibitor III; RNA; Ribose monophosphate abasic site.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Repair
DNA-(Apurinic or Apyrimidinic Site) Lyase* / analysis
DNA-(Apurinic or Apyrimidinic Site) Lyase* / metabolism
Enzyme Assays* / methods
In Vitro Techniques
Ribose / metabolism

Substances

DNA-(Apurinic or Apyrimidinic Site) Lyase
Ribose