A New Method for Exonuclease Activity Analysis of Apurinic/Apyrimidinic Endonuclease 1 and Application in Heavy-polluted Ramie

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1) exonuclease plays a vital proofreading role in the base excision repair pathway by removing 3’end group, including oxidatively damaged DNA bases, chain terminating nucleotide analog drugs, terminal blocking groups and mismatched bases. Herein, a simple and real time fluorometric method was developed for detecting APE1 exonuclease activity using molecular beacon as substrate. Experimental results demonstrated that the detection limit of this method was 0.01 U/μL for APE1, and the K m value was (0.27 ± 0.10) μmol/L. Moreover, the method was used to screen antibiotics and the results showed that streptomycin sulfate and gentamycin sulfate had a significant inhibitory effect on APE1 exonuclease activity in a concentration-dependent manner. Meanwhile, the assay was used for investigating the inhibitory effects of heavy metal ions on APE1 exonuclease activity. Finally, the method was further used to monitor the effect of heavy metal ions treatment of the APE1 exonuclease activity of ramie, and the results indicated that APE1 exonuclease activity was inhibited in a concentration-dependent manner after treatment with Pb 2+ and Cd 2+ , which was consistent with heavy metal ions assay in vitro . In summary, this method provided an alternative tool for the biochemical analysis for APE1 exonuclease activity, which was hopeful for the assessment for heavy metal ions adsorption according to the enzymatic activity change in phytoremediation. A convenient and sensitive real-time fluorometric method was proposed for APE1 exonuclease activity detection. This method performed well when it was applied for monitoring the effect of Pb 2+ and Cd 2+ treatment on APE1 exonuclease activity of ramie plant. This study provides an alternative tool for the biochemical analysis for APE1 exonuclease activity, which is hopeful for the assessment for heavy metals adsorption according to the enzymatic activity change in phytoremediation.