title: Novel method for screening of radioprotective agents providing protection to DNA ligase against gamma radiation induced damage.
abstract: OBJECTIVE
  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).
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Radiation Biotechnology Laboratory, Department of Radiation Biosciences.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Mishra
authors.forename: Poonam
authors.initials: P
authors.lastname: Malhotra
authors.forename: Ashutosh K
authors.initials: AK
authors.lastname: Gupta
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Singh
authors.forename: Anil K
authors.initials: AK
authors.lastname: Mishra
authors.forename: Saleem
authors.initials: S
authors.lastname: Javed
authors.forename: Raj
authors.initials: R
authors.lastname: Kumar
mesh_terms: D011088:DNA Ligases / Q000502:physiology / Q000528:radiation effects*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005720:Gamma Rays; D011837:Radiation-Protective Agents / Q000494:pharmacology*
chemical_list: D011837:Radiation-Protective Agents; D011088:DNA Ligases