Anchored linear oligonucleotides: the effective tool for the real-time measurement of uracil DNA glycosylase activity.

Anna Ligasová,Ivan Rosenberg,Markéta Bocková,Jiří Homola,Karel Koberna

doi:10.1098/rsob.210136

Abstract

Base excision repair is one of the important DNA repair mechanisms in cells. The fundamental role in this complex process is played by DNA glycosylases. Here, we present a novel approach for the real-time measurement of uracil DNA glycosylase activity, which employs selected oligonucleotides immobilized on the surface of magnetic nanoparticles and Förster resonance energy transfer. We also show that the approach can be performed by surface plasmon resonance sensor technology. We demonstrate that the immobilization of oligonucleotides provides much more reliable data than the free oligonucleotides including molecular beacons. Moreover, our results show that the method provides the possibility to address the relationship between the efficiency of uracil DNA glycosylase activity and the arrangement of the used oligonucleotide probes. For instance, the introduction of the nick into oligonucleotide containing the target base (uracil) resulted in the substantial decrease of uracil DNA glycosylase activity of both the bacterial glycosylase and glycosylases naturally present in nuclear lysates.

Highlights

DNA receives endogenous and exogenous insults every minute and the lesions are extremely deleterious to cells [1,2]
A similar ratio between the amount of UNG and nuclear and cytoplasmic lysates independently of the method of preparation was observed. These results indicated that the approach based on Triton X-100 is the preferential choice for the measurement of UNG1 activity while the method based on Dounce homogenization is preferential for the measurement of 14 UNG2 activity
We described two systems for the realtime measurement of glycosylase activity

Summary

Introduction

DNA receives endogenous and exogenous insults every minute (approx. 104–105 per cell per day) and the lesions are extremely deleterious to cells [1,2]. 104–105 per cell per day) and the lesions are extremely deleterious to cells [1,2]. These insults involve the action of chemical compounds and various forms of radiation. The loss of repair fidelity can be exploited for the treatment of cancer. In this respect, targeting the DNA repair has been an attractive strategy to overwhelm cancer cells with DNA damage, improve the efficacy of radiotherapy and/or chemotherapy, or form part of a lethal combination with a cancer-specific mutation/loss of function [7]

Methods

Results

Conclusion