Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase

Abstract

A coliphage M13 chimer containing the Saccharomyces cerevisiae TRPI gene and ARSJ replication origin (mPY2) was grown on an ung-dut-strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a Atrpl yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing singlestranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ungi. Strains containing the ungi mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNAglycosylase activity was demonstrated in the mutant strains.Uracil is introduced into DNA either during DNA replication by incorporation of dUMP instead of dTMP or by spontaneous or induced deamination of cytosine to uracil (10,19,28,30,33,39). From both of these modifications, although different in nature (i.e., a dA-dU base pair as a result of misincorporation versus a dG. dU base pair through deamination) uracil is efficiently excised by the enzyme uracil-DNA-glycosylase. This enzyme has been isolated from a wide variety of procaryotic and eucaryotic organisms (16). It is typically a monomeric enzyme with a molecular weight of 20,000 to 30,000, equally active on single-stranded (ss) and double-stranded (ds) DNAs (16). Unlike nucleases, which require Mg2+ for activity, uracil-DNA-glycosylase is fully active in the presence of EDTA. This facilitates activity measurements in crude extracts containing nucleases.Escherichia coli mutants (ung) deficient for uracil-DNAglycosylase have been useful in the study of the mechanism of DNA replication (24, 39) and in delineating excision repair pathways (36). Similarly, such a mutant in the yeast Saccharomyces cerevisiae could help in the study of analogous problems. To our knowledge all uracil-DNA-glycosylase mutants have been obtained by enzymatic screening of extracts from large numbers of colonies after mutagenesis. This approach, however, tends to generate predominantly leaky mutants, as indeed has been observed for E. coli, Bacillus subtilis, and the smut fungus Ustilago maydis (13,29,42). In addition, in vivo enzymatic activities may be different from those measured in vitro.The observation by Duncan et al. (13), however, that uracil-containing T4 phage will plate efficiently only on E. coli ung mutants with an in vitro enzymatic activity of <1% of that of the wild type led us to devise a genetic scheme to isolate uracil-DNA-glycosylase mutants in S. cerevisiae. Using a genetic transformation procedure, we selected for survival of the transforming D...

The Role of the Escherichia coli Mug Protein in the Removal of Uracil and 3,N 4-Ethenocytosine from DNA

“…The level of soluble Mug did not increase at higher concentrations of the inducer, probably because Mug tends to aggregate at high concentrations. 2 The cell viability is also low at concentrations above 40 M. Consequently, the assays were done at 30 M IPTG.…”

DNA glycosylases in the base excision repair of DNA

“…Mutational inactivation of the nuclear yeast UDG without affecting mitochondrial UDG activity [66], as well as different biochemical properties [61], have indicated that these enzymes are encoded by different genes. However, early studies demonstrated that the two forms are inhibited to the same extent by the very selective protein inhibitor Ugi.…”

ATP Utilization by Yeast Replication Factor C

“…pBL642 (RFC1, TRP1) was created by cloning the 5364-bp PvuI fragment from pBL641 into the 2871-bp PvuI fragment of pRS314. pBL642-R and pBL642-E (rfc1-K359R or rfc1-K359E, TRP1) were created by annealing mutagenic primers (1K359R, 5Ј-CCTGGTATTGGGCGTACGACTGCTGCTC-3Ј; 1K359E, 5Ј-CCTGG-TATTGGGGAGACAACTGCTGCTC-3Ј; mutations are underlined) to single-stranded, circular, uracil-containing pBL642 DNA (20) and extending the primers with T7 DNA polymerase, followed by ligation. These extension reactions were transformed into E. coli strain XL1-Blue.…”