REPAIR OF UV‐DAMAGED INCOMING PLASMID DNA IN Saccharomyces cerevisiae

Abstract

A whole-cell transformation assay was used for the repair of UV-damaged plasmid DNA in highly transformable haploid strains of Saccharomyces cerevisiae having different repair capabilities. Six rad alleles were selected from the three epistasis groups: rad 1-1 and rad2-1 from the RAD3 group, rad6-1 and rad18-2 from the RAD6 group, and rad52-1 and rad54-1 from the RAD52 group. Cells carrying single, double and triple rad alleles were transformed to uracil prototrophy by centromeric plasmid DNA (YCp19) modified in vitro with UV (254 nm). Surviving fractions were calculated as the number of transformants at each fluence relative to the number of transformants with unirradiated plasmid DNA. The sensitivity of incoming DNA in single rad mutants shows that most repair is carried out by excision repair and a RAD18-dependent process. In the rad52-1 host, the sensitivity of incoming DNA was intermediate between those found in RAD+ and rad2-1 hosts, suggesting the involvement of a recombinational repair process. Non-epistatic interactions were observed between rad alleles belonging to different epistasis groups. This provides validation for the classification of the three epistasis groups concerning the repair of chromosomal DNA for UV-incoming DNA. In both rad1-1 rad6-1 and rad1-1 rad18-2 rad54-1 hosts, the mean fluence for one lethal event corresponds approximately to one pyrimidine dimer per plasmid molecule, indicating that they are absolute repairless hosts for incoming DNA. A comparison between cell and plasmid survival reveals that there are differences in the repairability of both chromosomal and incoming DNA. The large effect of rad6-1 mutation on cell survival and the small effect on incoming DNA suggest that, in the RAD+ strain, the RAD6 product may be essential for the repair processes which act on chromosomal DNA, but not for those which act on incoming DNA. It is proposed that in yeasts postreplication repair of incoming DNA is limited to supercoiled molecules with 1-2 pyrimidine dimers that can initiate replication.

Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways

“…Hence, the growth kinetics of each strain cultured with BLM were compared with the corresponding values of the control without BLM treatment. The observed DT values of all studied strains were in accordance with those reported previously (18). As described in Materials and Methods, results indicated the lack of suppression of the rad6-1 mutation.…”

“…Concerning interactions among repair processes (8,9,12,13,18), our results showed epistasis between the RAD6 and RADS2 genes at low BLM concentrations. In fact, according to results obtained by other authors, RAD6 functions represent an earlier step required for repair, mutagenesis, recombination, and meiosis (16,21,41).…”