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 rnr4Δ mutant that allows only very little mutation induction at very low doses of 254nm 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 rnr4Δ 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 rnr4Δ 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.
Similar content being viewed by others
Abbreviations
- RNR:
-
Ribonucleotide reductase
- HU:
-
Hydroxyurea
- BM:
-
Benomyl
- WT:
-
Wild type
- UVC:
-
254nm ultraviolet light
- PUVA:
-
Photoactivated psoralen
- Y1/Y3:
-
Yeast RNR large subunits
- Y2/Y4:
-
RNR small subunits
- STAT:
-
Stationary growth phase
- CS:
-
Cold sensitivity
References
An X, Zhang Z, Yang K, Huang M (2006) Cotransport of the heterodimenric small subunit of the Saccharomyces cerevisiae ribonucleotide reductase between the nucleus and the cytoplasm. Genetics 173:63–73
Ausubel FM, Brent R, Kingston RE, Moore DD, Smith JA, Seideman JG, Struhl K (1989) Current protocols in molecular biology. Wiley, New York
Barclay BJ, Little JG (1981) Mutation induction in yeast by deoxythimidine monophosphate: a model. Mol Gen Genet 181:279–281
Bender E, Brendel M (1988) Effects of excess thymidylate on thymidylate low-requiring strains of Saccharomyces cerevisiae: high mutagenicity and absence of DNA strand breaks. Mutat Res 197:59–66
Brendel M (1985) Mutation induction by excess deoxyribonucleotides in Saccharomyces cerevisiae. In: de Serres FJ (ed) From: genetic consequences of nucleotide pool imbalance. Plenum Press, New York, pp 425–434
Brendel M, Grey M, Maris AF, Hietkamp J, Fesus F, Pich CT, Dafré L, Schmidt M, Eckardt-Schupp F, Henriques JAP (1998) Low glutathione pools in the original pso3 mutant of Saccharomyces cerevisiae are responsible for its pleiotropic sensitivity phenotype. Curr Genet 33:4–9
Brendel M, Henriques JAP (2001) The pso mutants of Saccharomyces cerevisiae comprise two groups: one deficient in DNA repair and another with altered mutagen metabolism. Mutat Res 489:79–96
Brendel M, Bonatto D, Strauss M, Revers LF, Pungartnik C, Saffi J, Henriques JAP (2003) Role of PSO genes in repair of DNA damage of Saccharomyces cerevisiae. Mutat Res 544:179–193
Cassier C, Chanet R, Henriques JAP, Moustacchi E (1980) The effects of the three PSO genes on induced mutagenesis: a novel class of mutationally defective yeast. Genetics 96:841–857
Cassier C, Moustacchi E (1981) Mutagenesis induced by mono- and bifunctional alkylating agents in yeast mutants sensitive to photo-addition of furocoumarins (pso). Mutat Res 84:37–47
Chabes A, Domkin V, Larsson G, Liu A, Gräslund A, Wijmenga S (2000) Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit. Proc Natl Acad Sci USA 97:2474–2479
Chabes A, Georgieva B, Domkin V, Zhao X, Rothstein R, Thelander L (2003) Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase. Cell 112:391–401
Elledge SJ, Zhou Z, Allen JB (1992) Ribonucleotide reductase: regulation, regulation, regulation. Trends Biochem Sci 17:119–123
Elledge SJ, Zhou Z, Allen JB, Navas TA (1993) DNA damage and cell cycle regulation of ribonucleotide reductase. Bioessays 15:333–339
Ge J, Perlstein DL, Nguyen HH, Bar G, Griffin RG, Stubbe J (2001) Why multiple small subunits (Y2 and Y4) for yeast ribonucleotide reductase? Toward understanding the role of Y4. Proc Natl Acad Sci USA 98:10067–10072
Haracska L, Prakask S, Prakash L (2003) Yeast DNA polymerase zeta is an efficient extender of primer ends opposite from 7,8-dihydro-8-oxoguanine and O6-methylguanine. Mol Cell Biol 23:1453–1459
Henriques JAP, Moustacchi E (1980) Isolation and characterization of pso mutants sensitive to photoaddition of psoralen derivatives in Saccharomyces cerevisiae. Genetics 95:273–288
Henriques JAP, Brozmanova J, Brendel M (1997) Role of PSO genes in the repair of photoinduced interstrand cross-links and photooxidative damage in the DNA of the yeast Saccharomyces cerevisiae. J Photochem Photobiol B 39:185–196
Huang M, Elledge SJ (1997) Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae. Mol Cell Biol 17:6105–6113
Koc A, Wheeler LJ, Mathews CK, Merrill GF (2003) Replication-independent MCB gene induction and deoxyribonucleotide accumulation at G1/S in Saccharomyces cerevisiae. J Biol Chem 278:9345–9352
Lemontt J (1971) Mutants of yeast defective in mutation induced by ultraviolet light. Genetics 68:21–33
Nguyen HH, Ge J, Perlstein DL, Stubbe J (1999) Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly. Proc Natl Acad Sci USA 96:12339–12344
Pungartnik C, Kern MC, Brendel M, Henriques JAP (1999) Mutant allele pso7-1, that sensitises Saccharomyces cerevisiae to photoactivated psoralen, is allelic with COX11, encoding a protein indispensable for a functional cytochrome c oxidase. Curr Genet 36:124–129
Pungartnik C, Picada J, Brendel M, Henriques JAP (2002) Further phenotypic characterization of pso mutants of Saccharomyces cerevisiae with respect to DNA repair and response to oxidative stress. Genet Mol Res 1:79–89
Rose MD, Novick P, Thomas JH, Botstein D, Fink G (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromer-containing shuttle vector. Gene 60:237–243
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. CSH Laboratory Press, Cold Spring Harbor, New York
Schmidt CL, Grey M, Schmidt M, Brendel M, Henriques JAP (1999) Allelism of Saccharomyces cerevisiae genes PSO6, involved in survival after 3-CPs+UVA induced damage, and ERG3, encoding the enzyme sterol C-5 desaturase. Yeast 15:1503–1510
Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27
Wang PJ, Chabes A, Casagrande R, Tian XC, Thelander L, Huffaker TC (1997) Rnr4p, a novel ribonucleotide reductase small-subunit protein. Mol Cell Biol 17:6114–6121
Yao R, Zhang Z, An X, Bucci B, Perlstein DL, Stubbe J, Huang M (2003) Subcellular localization of yeast ribonucleotide reductase regulated by the DNA replication and damage checkpoint pathways. Proc Natl Acad Sci USA 100:6628–6633
Zhao X, Rothstein R (2002) The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1. Proc Natl Acad Sci USA 99:3746–3751
Acknowledgments
We wish to thank Dr. Diego Bonatto for designing Fig. 7. M.S. held a fellowship of the Hessische Graduiertenfoerderung. M.B. and J.A.P.H. were supported by travel grants from DAAD-CNPq. M.B. is now a Visiting Scientist at UESC sponsored by the Fundação de Amparo a Pesquisa do Estado do Bahia (FAPESB).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by J. Hasek.
Rights and permissions
About this article
Cite this article
Strauss, M., Grey, M., Henriques, J.A.P. et al. RNR4 mutant alleles pso3-1 and rnr4Δ block induced mutation in Saccharomyces cerevisiae . Curr Genet 51, 221–231 (2007). https://doi.org/10.1007/s00294-007-0120-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-007-0120-7