Kinetics of Uv-Induced Gene and Structural Mutations

Abstract

Abstract. To study the kinetics of UV-induced gene and chromosome mutations in the yeast S. cerevisiae, several genetic assays were used. We treated yeast cells with UV light of up to 130 J/m 2 . UV irradiation induced all types of base substitutions, although transitions -in particular, GC-AT events -were predominant. Frameshift mutations were induced at the same frequency as the base pair substitution GC-AT, while forward mutations in the CAN1 gene exceeded the more expressive base pair substitutions by about an order of magnitude. Chromosome mutations were the most efficient. The kinetic of the induced gene and chromosome mutations is represented by a linear-quadratic function. Such curves have been reported for UV mutagenesis in bacteria and they have been explained by the induction of SOS error-prone repair. Similar biphasic kinetics was described for yeast in our work. These data suggest the occurrence of several factors forming the mutagenic response of eukaryotic cells to UV light.Key words: UV irradiation, frameshift mutation, base pair substitution, forward mutation, rearrangement of chromosome and plasmid DNA, yeast Ultraviolet (UV) light has strong genotoxic effectsit can induce DNA damage, mutations, and, in the worst case, cancerogenesis. Several human genetic disorders, including xeroderma pigmentosum and Cockayne syndrome, are characterized by a defect in UV lesion repair. Investigations of the mechanisms of mutagenesis are continued and our knowledge is made more profound. To study the kinetics of the UVinduced gene and structural mutations on the bases of the model of the yeast S. cerevisiae, several new interesting genetic assays were used. They included a forward mutation rate assay that detects mutations inactivating the arginine permease gene (Can R mutations), frameshift reversion assays [1] detecting mutation that reverts a 4-base insertion in the LYS2 gene (lys2ΔBgl), and a collection of six isogenic trp5-strains, specifically diagnostic for all possible base-pair substitutions [2]. Assays for intrachromosome homologous recombination (HR repair) based on the 5'trancated lys2 sequence and the LEU2 gene integrated into chromosome II as a direct repeat with the lys2:HS-D allele which is 658 bp-insertion in BamH1 site of 3'-termini of LYS2 gene are shown in [3]. The [YCpL2]-plasmid assay was used to detect the extent of deletions including two or more genes which arise during NHEJ repair [4].Mutant strains were grown nonselectively in YEPD. Selective growth was on a synthetic complete medium containing 2 % glucose (SM) [5] and lacking the appropriate nutrient [6]. Canavanine-resistant mutants in the forward mutation assay were identified on SM-Arg plates supplemented with 60 μg/ml canavanine. All growth was at 30 o C. Overnight cultures (~2x10 8 cells/ml) were grown in 5 ml YEPD. Cells were resuspended in water and plated on YEPD and appropriate SM-based selective media to assess cell survival and mutagenesis, respectively. Within 1 hr of plating, cells were exposed to 254 nm UV light and va...