RNR4 mutant alleles pso3-1 and rnr4Δ block induced mutation in Saccharomyces cerevisiae

Abstract

The PSO3 gene of Saccharomyces cerevisiae was molecularly cloned by complementing the cold-sensitivity phenotype of a pso3-1 mutant and was found to be allelic to RNR4, encoding one of the two DNA damage-inducible small subunits of the ribonucleotide reductase (RNR) complex. Compared to a rnr4Delta mutant that allows only very little mutation induction at very low doses of 254(nm) ultraviolet light (UVC), the pso3-1 mutant allele confers leakiness in that it permits some DNA damage-induced mutagenesis at low doses of UVC. Similarly, the pso3 mutant is slightly less sensitive to UVC than an rnr4Delta mutant. Cloning and sequencing of the RNR4 locus of the pso3-1 mutant revealed that its intermediate phenotype is attributable to a G --> A transition at nucleotide 352, leading to replacement of glycine by arginine [G118R] in the mutant's protein. Both RNR4 mutant alleles confer significantly less sensitivity to UVC than mutant alleles of non-UVC-mutable REV3, indicating that, apart from nucleotide excision repair, RAD6-dependent error-free DNA repair may still be functional. The phenotype of a strongly reduced UVC-induced mutagenesis for rnr4 mutant alleles has not yet been described; it suggests the importance of this gene for a fully functional RNR providing correct amounts of DNA precursor molecules, thereby, allowing translesion synthesis (error-prone) of UVC-damaged DNA. Stationary phase cells of the rnr4Delta mutant, but not of the original pso3-1 mutant, are swollen with a fourfold to eightfold increase in volume. The central role of RNR in DNA precursor metabolism and its complex regulation allow for several modes of suppression that may influence the phenotypes of RNR4 mutants, especially those containing the leaky pso3-1 mutant allele.

Identification of pathways controlling DNA damage induced mutation in Saccharomyces cerevisiae

“…These data suggest that the effect on induced mutation may be generalized to other subunits of the RNR complex. We also demonstrated that RNR4 is required for UV-and MMS-induced increases in dNTP levels, consistent with the ability of the protein to stimulate TLS as proposed previously [38]. Moreover, our data suggest that RNR4 functions in a previously unknown second pathway of induced mutation that involves TLS by the replicative polymerase Polδ.…”

“…Previously, Brendel and co-workers demonstrated that deletion of RNR4 reduces UV-induced mutation and the authors suggested that this resulted from reduced translesion synthesis by Polζ [38]. We first examined the UV sensitivity and mutability of a rad1Δ rnr4Δ mutant to determine whether Rnr4 is involved in NER, the primary repair mechanism responsible for removing UV-lesions.…”

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride

“…The lower processivity of RNR in an rnr4Δ mutant (Y1Y3Y2Y2) that is due to the replacement of the small subunit heterodimer Y2Y4 by the homodimer Y2Y2 leaves the cell with significantly lower-than-normal dNTP pools (Perlstein et al, 2005). These nonadequate dNTP pools lead to somewhat slower cell growth and to significantly increased UVC sensitivity apparently by not allowing efficient repair of DNA damage in rnr4Δ (Strauss et al, 2007) and in the mutant harboring the leaky RNR4 mutant allele pso3-1 Cassier et al, 1980;Brendel et al, 1998). The observed sensitivity to SnCl 2 of the mutant rnr4Δ may thus reflect the (partial) loss of capacity to repair DNA damage.…”

“…Imbalanced dNTP pools with one (Brendel, 1985) or all (Chabes et al, 2003) dNTPs at higher-than-normal concentrations lead to increased mutation rates, while an rnr4Δ mutant with a Y2Y2 small subunit homodimer that contains 15 times less diferric-tyrosyl radical co-factor (Perlstein et al, 2005) and apparently lower dNTP pools exhibits slow growth, increased mutagen sensitivity and lower induced mutagenesis (Strauss et al, 2007).…”

“…The facultative anaerobe yeast Saccharomyces cerevisiae can grow in the presence or absence of respiratory metabolism in glucose-containing cultures (sugar fermentation, followed by alcohol respiratory metabolism after the diauxic shift); therefore, cells of different stages of the same culture serve as a good model to test the influence of general metabolism on sensitivity to Sn 2+ (Viau et al, 2006). Since Strauss et al (2007) have shown that the rnr4Δ mutant strain has a higher-than-wild-type (WT) budding index, and exponentially growing (LOG) cells are known to be much more sensitive to SnCl 2 exposure (Viau et al, 2006), we hypothesized that the lack of fully functional RNR would leave the cells in a LOG-type growth phase and more sensitive to SnCl 2 .…”