DNA sequence analysis of spontaneous mutations in the SUP4-o gene of Saccharomyces cerevisiae.

A collection of 196 spontaneous mutations in the SUP4-o gene of the yeast Saccharomyces cerevisiae was analyzed by DNA sequencing. The classes of mutation identified included all possible types of base-pair substitution, deletions of various lengths, complex alterations involving multiple changes, and insertions of transposable elements. Our findings demonstrate that at least several different mechanisms are responsible for spontaneous mutagenesis in S. cerevisiae.     

Deletion analysis of the SUP2 gene in Saccharomyces cerevisiae

The sup2 mutations of the yeast Saccharomyces cerevisiae or plasmid-mediated amplification of the wild type SUP2 gene lead to suppression of different types of nonsense mutations. The Sup2 protein includes a C-terminal region homologous to elongation factor EF-1 alpha and an unique N-terminal region. The SUP2 is an essential gene. The functional role of different regions of the SUP2 gene was investigated, by deleting them without disruption of the reading frame. Such constructs were maintained in yeast on episomal or centromeric plasmids. It was shown that the region, homologous to EF-1 alpha is necessary for viability, while the remaining N-terminal part is nonessential. The region of the first 154 amino acids is necessary and sufficient for the suppressor effect, caused by plasmid-mediated amplification of the SUP2 gene.     

Influence of neighbouring base sequence on N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the lacI gene of Escherichia coli

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations within the first 540 base-pairs of the lacI gene of Escherichia coli were cloned and sequenced. In total, 167 MNNG-induced independent mutations were characterized, with G.C to A.T transitions accounting for all but three of the mutations. This mutagenic specificity is consistent with the mispairing predicted by the methylation of the O6 position of guanine. The characterization of such large numbers of mutations permitted an analysis of the influence of local DNA sequence on mutagenesis. This analysis revealed a strong influence by the 5' flanking base. On average, guanine residues preceded (5') by a guanine or an adenine residue were, respectively, nine times and five times more likely to mutate after treatment with MNNG than those preceded by a pyrimidine residue.     

Interaction of ATP17 gene with SUP45 and SUP35 genes in Saccharomyces cerevisiae yeast

Genes SUP35 and SUP45 have been identified in the saccharomycete yeast as genes controlling termination of translation in cytoplasmic ribosomes. However, many facts indicate that the control of translation termination is not the only function of these genes. This work is devoted to studying one of the pleiotropic effects of sup35 and sup45 mutations, a respiratory deficiency. The compensation for this deficiency in mutants for either gene can occur due to a mutation in the ATP17 gene encoding the f-subunit of mitochondrial F1F0 ATP synthase. It is assumed that the observed interaction can be related to the system of co-translational protein import into mitochondria.     

Polymorphism of the SUP35 gene and its product in the Saccharomyces cerevisiae yeasts

The product of the SUP35 gene of the saccharomycete yeast, the translation termination eRF3 factor, can be converted in prion, the heritable determinant of protein nature. The nucleotide sequence of this gene from the strain belonging to Peterhof genetic lines of the yeast Saccharomyces cerevisiae was determined. A comparison of the identified sequence with SUP35 sequences in the database of GenBank allowed the detection of polymorphic sites both in the SUP35 gene and its product. The location of polymorphic sites in the evolutionarily nonconserved N-terminal protein region confirmed that this eRF3 fragment lacks functions vital to life activity. Nevertheless, these sites are located in the vicinity of sites, whose role in the prion conversion of eRF3 has been established. Based on this, natural polymorphism of the primary eRF3 structure is assumed to be connected with the existence of different variants (strains) of its prion analog.     

Base-pair substitutions alter the site-specific mutagenicity of UV and MNNG in the SUP4-o gene of yeast

Yeast strains carrying SUP4-o genes that have base-pair substitutions at hotspots for UV or MNNG mutagenesis were treated with these agents. In both cases, the induced mutation frequencies were substantially reduced. Furthermore, specific substitutions at positions in SUP4-o that had not been mutated by MNNG resulted in the recovery of MNNG-induced mutations at these sites. These results demonstrate that base-pair identity is an important factor determining the site-specific mutagenicity of UV and MNNG in yeast. For UV, our findings suggest that the type of lesion that is induced, but not flanking DNA sequences, plays a role in specifying mutability at the sites examined. In contrast, DNA sequence context seems to be an important factor for MNNG mutagenesis.     

Prionization of the Pichia methanolica SUP35 gene product in the yeast Saccharomyces cerevisiae

Induction of the prionlike form of the SUP35 gene of Pichia methanolica, the [PSIP+] factor, was shown in the transgenic yeast Saccharomyces cerevisiae containing the P. methanolica SUP35 gene located in the chromosome instead of the indigenous SUP35 gene. Either the induction of the [PSIP+] factor in the transgenic yeast, unlike that of the classical [PSI+] factor, does not depend on the presence of the [PIN+] determinant in the cell or the substitution of the S. cerevisiae SUP35 gene for the P. methanolica SUP35 gene changes the PIN status of the strain. The [PSIP+] factor is unstable in mitosis and meiosis and is not effectively eliminated upon over-production of the chaperone protein Hsp104p of S. cerevisiae. The existence of an interspecific barrier during transmission of the prionlike state from S. cerevisiae Sup35p to P. methanolica Sup35p was shown.     

Substrate specificity of the Saccharomyces cerevisiae Mus81-Mms4 endonuclease

Mus81-Mms4/Eme1 is a conserved structure-specific endonuclease that functions in mitotic and meiotic recombination. It has been difficult to identify a single preferred substrate of this nuclease because it is active on a variety of DNA structures. In addition, it has been suggested that the specificity of the recombinant protein may differ from that of the native enzyme. Here, we addressed these issues with respect to Mus81-Mms4 from S. cerevisiae. At low substrate concentrations, Mus81-Mms4 was active on any substrate containing a free end adjacent to the branchpoint. This includes 3'-flap (3'F), regressed leading strand replication fork (RLe), regressed lagging strand replication fork (RLa), and nicked Holliday junction (nHJ) substrates. Kinetic analysis was used to quantitate differences between substrates. High Kcat/Km values were obtained only for substrates with a 5'-end near the branchpoint (i.e., 3'F, RLe, and nHJ); 10-fold lower values were obtained for nicked duplex (nD) and RLa substrates. Substrates lacking any free ends at the branch point generated Kcat/Km values that were four orders of magnitude lower than those of the preferred substrates. Native Mus81-Mms4 was partially purified from yeast cells and found to retain its preference for 3'F over intact HJ substrates. Taken together, these results narrow the range of optimal substrates for Mus81-Mms4 and indicate that, at least for S. cerevisae, the native and recombinant enzymes display similar substrate specificities.     

Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae

To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.     

Characterization of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1

Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1-eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.     

Viable nonsense mutants for the SUP45 gene in the yeast Saccharomyces cerevisiae are lethal at increased temperature

Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination eRFI factor in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a truncated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37 degrees C. When grown under nonpermissive conditions (37 degrees C), cells of nonsense mutants sup45-104, sup45-105. and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.     

DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae.

DNA postreplication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes. In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities. PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown. This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes. Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed. Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle. The PRR processes appear to be highly conserved within eukaryotes, from yeast to human.