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. 1997 Oct;17(10):6114–6121. doi: 10.1128/mcb.17.10.6114

Rnr4p, a novel ribonucleotide reductase small-subunit protein.

P J Wang 1, A Chabes 1, R Casagrande 1, X C Tian 1, L Thelander 1, T C Huffaker 1
PMCID: PMC232461  PMID: 9315671

Abstract

Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex.

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Selected References

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  1. Bond J. F., Fridovich-Keil J. L., Pillus L., Mulligan R. C., Solomon F. A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo. Cell. 1986 Feb 14;44(3):461–468. doi: 10.1016/0092-8674(86)90467-8. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  4. Christianson T. W., Sikorski R. S., Dante M., Shero J. H., Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119–122. doi: 10.1016/0378-1119(92)90454-w. [DOI] [PubMed] [Google Scholar]
  5. Deisenhofer J., Michel H. Structures of bacterial photosynthetic reaction centers. Annu Rev Cell Biol. 1991;7:1–23. doi: 10.1146/annurev.cb.07.110191.000245. [DOI] [PubMed] [Google Scholar]
  6. Eichinger L., Bomblies L., Vandekerckhove J., Schleicher M., Gettemans J. A novel type of protein kinase phosphorylates actin in the actin-fragmin complex. EMBO J. 1996 Oct 15;15(20):5547–5556. [PMC free article] [PubMed] [Google Scholar]
  7. Elledge S. J., Davis R. W. Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability. Mol Cell Biol. 1987 Aug;7(8):2783–2793. doi: 10.1128/mcb.7.8.2783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elledge S. J., Davis R. W. Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase. Genes Dev. 1990 May;4(5):740–751. doi: 10.1101/gad.4.5.740. [DOI] [PubMed] [Google Scholar]
  9. Engström Y., Eriksson S., Thelander L., Akerman M. Ribonucleotide reductase from calf thymus. Purification and properties. Biochemistry. 1979 Jul 10;18(14):2941–2948. doi: 10.1021/bi00581a004. [DOI] [PubMed] [Google Scholar]
  10. Fontecave M., Nordlund P., Eklund H., Reichard P. The redox centers of ribonucleotide reductase of Escherichia coli. Adv Enzymol Relat Areas Mol Biol. 1992;65:147–183. doi: 10.1002/9780470123119.ch4. [DOI] [PubMed] [Google Scholar]
  11. Fox T. D., Folley L. S., Mulero J. J., McMullin T. W., Thorsness P. E., Hedin L. O., Costanzo M. C. Analysis and manipulation of yeast mitochondrial genes. Methods Enzymol. 1991;194:149–165. doi: 10.1016/0076-6879(91)94013-3. [DOI] [PubMed] [Google Scholar]
  12. Huang M., Elledge S. J. Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae. Mol Cell Biol. 1997 Oct;17(10):6105–6113. doi: 10.1128/mcb.17.10.6105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hurd H. K., Roberts C. W., Roberts J. W. Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate. Mol Cell Biol. 1987 Oct;7(10):3673–3677. doi: 10.1128/mcb.7.10.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hurt E. C. A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae. EMBO J. 1988 Dec 20;7(13):4323–4334. doi: 10.1002/j.1460-2075.1988.tb03331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hutter K. J., Eipel H. E. Microbial determinations by flow cytometry. J Gen Microbiol. 1979 Aug;113(2):369–375. doi: 10.1099/00221287-113-2-369. [DOI] [PubMed] [Google Scholar]
  16. Jacobo-Molina A., Ding J., Nanni R. G., Clark A. D., Jr, Lu X., Tantillo C., Williams R. L., Kamer G., Ferris A. L., Clark P. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6320–6324. doi: 10.1073/pnas.90.13.6320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kauppi B., Nielsen B. B., Ramaswamy S., Larsen I. K., Thelander M., Thelander L., Eklund H. The three-dimensional structure of mammalian ribonucleotide reductase protein R2 reveals a more-accessible iron-radical site than Escherichia coli R2. J Mol Biol. 1996 Oct 11;262(5):706–720. doi: 10.1006/jmbi.1996.0546. [DOI] [PubMed] [Google Scholar]
  18. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  19. Krakoff I. H., Brown N. C., Reichard P. Inhibition of ribonucleoside diphosphate reductase by hydroxyurea. Cancer Res. 1968 Aug;28(8):1559–1565. [PubMed] [Google Scholar]
  20. Lowdon M., Vitols E. Ribonucleotide reductase activity during the cell cycle of Saccharomyces cerevisiae. Arch Biochem Biophys. 1973 Sep;158(1):177–184. doi: 10.1016/0003-9861(73)90611-5. [DOI] [PubMed] [Google Scholar]
  21. Mann G. J., Gräslund A., Ochiai E., Ingemarson R., Thelander L. Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit. Biochemistry. 1991 Feb 19;30(7):1939–1947. doi: 10.1021/bi00221a030. [DOI] [PubMed] [Google Scholar]
  22. Nordlund P., Eklund H. Structure and function of the Escherichia coli ribonucleotide reductase protein R2. J Mol Biol. 1993 Jul 5;232(1):123–164. doi: 10.1006/jmbi.1993.1374. [DOI] [PubMed] [Google Scholar]
  23. Pasqualone D., Huffaker T. C. STU1, a suppressor of a beta-tubulin mutation, encodes a novel and essential component of the yeast mitotic spindle. J Cell Biol. 1994 Dec;127(6 Pt 2):1973–1984. doi: 10.1083/jcb.127.6.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reichard P. From RNA to DNA, why so many ribonucleotide reductases? Science. 1993 Jun 18;260(5115):1773–1777. doi: 10.1126/science.8511586. [DOI] [PubMed] [Google Scholar]
  25. Riles L., Dutchik J. E., Baktha A., McCauley B. K., Thayer E. C., Leckie M. P., Braden V. V., Depke J. E., Olson M. V. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. doi: 10.1093/genetics/134.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  27. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  28. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  29. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stubbe J. Ribonucleotide reductases. Adv Enzymol Relat Areas Mol Biol. 1990;63:349–419. doi: 10.1002/9780470123096.ch6. [DOI] [PubMed] [Google Scholar]
  31. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  32. Thomas J. H., Neff N. F., Botstein D. Isolation and characterization of mutations in the beta-tubulin gene of Saccharomyces cerevisiae. Genetics. 1985 Dec;111(4):715–734. doi: 10.1093/genetics/111.4.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vitols E., Bauer V. A., Stanbrough E. C. Ribonucleotide reductase from Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1970 Oct 9;41(1):71–77. doi: 10.1016/0006-291x(70)90470-5. [DOI] [PubMed] [Google Scholar]

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