RESUMO
In order to extend the understanding of the genetical and biochemical basis of photo-activated psoralen-induced DNA repair in the yeast Saccharomyces cerevisiae we have identified and cloned 10 pso mutants. Here, we describe the phenotypic characterization and molecular cloning of the pso10-1 mutant which is highly sensitive to photoactivated psoralens, UV(254) (nm) radiation and the alkylating agent methylmethane sulphonate. The pso10-1 mutant allele also confers a block in the mutagenic response to photoactivated psoralens and UV(254) (nm) radiation, and homoallelic diploids do not sporulate. Molecular cloning using a yeast genomic library, sequence analysis and genetic complementation experiments proved pso10-1 to be a mutant allele of gene MMS21 that encodes a SUMO ligase involved in the sumoylation of several DNA repair proteins. The ORF of pso10-1 contains a single nucleotide C-->T transition at position 758, which leads to a change in amino acid sequence from serine to phenylalanine [S253F]. Pso10-1p defines a leaky mutant phenotype of the essential MMS21 gene, and as member of the Smc5-Smc6 complex, still has some essential functions that allow survival of the mutant. DNA repair via translesion synthesis is severely impaired as the pso10-1 mutant allele confers severely blocked induced forward and reverse mutagenesis and shows epistatic interaction with a rev3Delta mutant allele. By identifying the allelism of PSO10 and MMS21 we demonstrate the need of a fully functional Smc5-Smc6 complex for a WT-like adequate repair of photoactivated psoralen-induced DNA damage in yeast.
