MO_III_L: Molecular Sciences III. Lectures
dr. Pasitka Jonatán, Biophysics Department/University of Pécs, Pécs
Dr. Lukásc András, Biophysics Department/University of Pécs, Pécs
Introduction. The E.coli photolyase is an enzyme that is responsible for the repair of UV-light induced cyclobutane-pyrimidine (CPD) DNA lesions. Previously we have measured the ultrafast absorptive characteristics of different E.coli photolyase variants, namely the E109A mutation containing wild-type-like (m4) and two distinct cryptochrome-like mutants, containing N378DE109A (m1m4) and N378CE109A (m4m5) mutations. The N378 is thought to be essential for the photorepair activity of photolyases.
Aims. The aim of this study was to determine whether the m1m4 and m4m5 photolyase mutants show any DNA photorepair activity and if they do, assess the magnitude of their repair capacity compared to the wild-type-like E109A photolyase mutant.
Method. The CPD substrates were generated with UV-irradiation (~312 nm) of pdT18 primers for 6 hours. The thymidine shows great absorption at 265 nm. The formation of the CPD lesions could be monitored by the decreasing absorption of the pdT18 sample at this given wavelength. The photorepair activity assay contained 27 µM enzyme, 13 µM CPD in a 10 mM phosphate-buffer with 200 mM NaCl. The photorepair activity of the E.coli photolyase mutants were also assessed in the presence of 1 mM L-cysteine. The measurements were performed on an Ocean Optics STS-UV spectrophotometer. The indicator of the photorepair activity was the increasing absorption peak at 265 nm due to the restoration of the integrity of the pdT18 chains.
Results .Not surprisingly, the m4 mutant showed the greatest photorepair activity (ΔA265 = 0.457 in 25 min without L-cyst.), even after being completely oxidized with imidazole (ΔA265 = 0.315 and ΔA265 = 0.339 in 25 min, with and without L-cyst., respectively), however, contrary to our previous expectations the m1m4 and m4m5 mutants showed photorepair activity (m1m4: ΔA265 = 0.238 and ΔA265 = 0.193 in 25 min; m4m5: ΔA265 = 0.278 and ΔA265 = 0.147 in 25 min, with and without L-cyst., respectively).
Conclusions. These results suggests that in vitro the N378 aminoacid is not the only significant electron donor of the FAD, because if it had been than the m1m4 and m4m5 mutants would have lost their photorepair activity.
Funding. Supported by the ÚNKP-20-3.-I. New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund.
University of Pécs Medical School, Doctoral School of Interdisciplinary Medical Sciences