Expression of the E.coli ada gene in S.cerevisiae provides cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine in rad6 but not in rad52 mutants.
The Escherichia coli ada gene protein coding region under the control of the yeast alcohol dehydrogenase promoter in the extrachromosomally replicating yeast expression vectors pADHO6C and pVT103LO6C was introduced into the wild-type yeast strains, YNN-27 and FF-18733, and the repair deficient mutants LN-1 (rad1-1), VV-5 (rad6-1), C5-6 (rad52-1) and FF-18742 (rad52::URA3). This resulted in the expression of 3950, 1900, 1870, 1620, 1320 and 1420 fmol ada-encoded ATase/mg protein respectively: transformation with the parent vectors resulted in ATase activities of 3-17 fmol/mg protein. The wild-types, rad1-1 and rad6-1 yeast expressing the bacterial ATase showed increased resistance to the toxic and mutagenic effects of N-methyl-N'-nitro-N- nitrosoguanidine (MNNG). Expression of ATase in the rad52-1 and rad52::URA3 mutants neither complemented their sensitivity, nor reduced the mutagenic effects of this agent. These results suggest that whilst a portion of the toxic and mutagenic lesions induced by MNNG can be repaired in yeast by the E.coli Ada protein in a RAD1- and RAD6-independent manner, the RAD52 gene product may be essential for the complete functioning of the Ada ATase. This is the first suggestion of a possible cofactor requirement for ATase.
Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava.
J
J
Brozmanová
V
V
Vlcková
M
M
Chovanec
L
L
Cernáková
M
M
Skorvaga
G P
GP
Margison
D001426:Bacterial Proteins / Q000502:physiology*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000502:physiology; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000502:physiology; D005798:Genes, Bacterial; D005800:Genes, Fungal; D008025:Ligases / Q000502:physiology; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D008780:Methyltransferases / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase; D051721:Rad52 DNA Repair and Recombination Protein; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D044763:Ubiquitin-Conjugating Enzymes
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008769:Methylnitronitrosoguanidine; D008780:Methyltransferases; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases