Detection of DNA oxidation and DNA alkylation in Chlamydomonas reinhardtii by the comet assay.: Different sensitivity of wild type and DNA repair mutants

We applied the modified comet assay to investigate different DNA lesions in the alga Chlamydomonas reinhardtii arising either spontaneously or after treatment with MMS and menandione. For this purpose the wild type (W1), and two repair-deficient strains of the C. reinhardtii were used. The mutant strain Uvs 12 is deficient in nuclear excision repair pathway, whereas mutant strain Uvs E1 is blocked in recombination repair. The sensitivity of strains to alkylation was studied with the alkylating agent MMS. Menadione was applied to induce oxidative DNA damage. To detect oxidative DNA lesions we used both endonuclease III (for oxidised pyrimidines and AP sites) and formamidopyrimidinglycosylase (for oxidised and ring-opened purines, and AP sites). DNA alkylation was detected with AlkA (3-methyladenine DNA glycosylase II). Both mutants were more sensitive than Wi for induction of strand breaks after MMS treatment. Using Alk A we detected a higher level of DNA alkylations in mutant Uvs Fl. MMS induced a low proportion of AP sites after MMS treatment in all three strains. Compared with wild type both mutants were more sensitive to DNA oxidation.