Novel method for screening of radioprotective agents providing protection to DNA ligase against gamma radiation induced damage

Abstract

Purpose: A simple, sensitive and novel method was developed to screen out potential agents able to protect functional activity of DNA ligase against gamma irradiation-induced damage. Repeatability, authenticity and sensitivity of the method was verified by analyzing DNA ligase protecting activities of well-known radioprotectors such as amifostine, trolox, melatonin, semiquinone glucoside derivative (SQGD) and an antioxidant gallic acid in extremely low concentration (1 μg/reaction).

Material and methods: Two different sets (Set A and B) of T4 DNA ligase (1 unit/set) were prepared. Set ‘A’ (negative control) was exposed to different doses (3–5 kGy) of gamma radiation in the absence of radioprotective compounds. Set B (test) was exposed to similar doses of gamma radiation in the presence of radioprotective compounds. Following irradiation, DNA ligase was mixed with λ DNA (250 ng) pre-digested with Hind III restriction endonuclease. Ligation reaction was performed in both sets simultaneously at 22°C for 20 min and reaction product was analyzed using agarose gel electrophoresis.

Results: Complete DNA ligation was observed in samples where DNA ligase was irradiated in the presence of radioprotectective compounds, i.e., amifostine, trolox, melatonin and a natural radioprotector semiquinone glucoside derivative (SQGD) individually, while, functional impairment in ligation activity of DNA ligase was evident in samples in which DNA ligase was irradiated in the absence of a radioprotective compound.

Conclusion: The current method was able to provide significant input to screen out radioprotective compounds able to protect DNA ligase functional activity against gamma radiation-induced functional impairment.

Keywords::

• T4 DNA ligase
• SQGD
• amifostine
• melatonin
• trolox
• gamma radiation

Acknowledgements

The authors would like to acknowledge Defence Research and Development Organization, India, for providing the financial support for the present study (Project No. 1.3 of ST-P1, INM-311) as well as for awarding Senior Research Fellowship. They would also like to acknowledge Dr Neeraj Satija, Senior Research Associate, for providing guidance in designing and conducting experiments.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.