POLI and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of ... Lehman) for 1 h at 4°C. The nitrocellulose was then washed five times with ...
MOLECULAR AND CELLULAR BIOLOGY, Aug. 1987, p. 2783-2793 0270-7306/87/082783-11$02.00/0 Copyright © 1987, American Society for Microbiology
Vol. 7, No. 8
Identification and Isolation of the Gene Encoding the Small Subunit of Ribonucleotide Reductase from Saccharomyces cerevisiae: DNA Damage-Inducible Gene Required for Mitotic Viability STEPHEN J. ELLEDGE* AND RONALD W. DAVIS Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 Received 16 January 1987/Accepted 4 May 1987 Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in Agtll by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POLI and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells. The precursors for DNA synthesis, deoxyribonucleotides, are produced by direct reduction of their corresponding ribonucleotides in all organisms thus far examined. With the exception of lactobacilli (21), the reaction is of the form: ribonucleoside diphosphate + reductant-(SH)2-* deoxyribonucleoside diphosphate + reductant-(S-S), and it is catalyzed by the enzyme ribonucleoside diphosphate reductase
subunit (11, 12). It is not known whether the degradation rate is modulated in a cell cycle-dependent fashion. The Ml protein is expressed at a constant level throughout the cell cycle. Another type of regulation is found in oocytes. The RNA encoding the small subunit protein in both clam and sea urchin eggs exists as a stored maternal mRNA that is translationally activated upon fertilization (34). There is no evidence of synthesis of the large subunit after fertilization, and it is assumed to be synthesized in advance. In contrast to the mammalian enzyme, little is known about the enzyme from Saccharomyces cerevisiae. Ribonucleotide reduction was investigated by Vitols et al. (39) and Lowden and Vitols (20), who observed an activity maximum during S phase which was inhibited by hydroxyurea. Attempts by Vitols and others (18) to purify the enzyme failed due to the apparent instability of the enzyme. In this paper we describe the isolation and characterization of the gene encoding the small subunit of ribonucleotide reductase from S. cerevisiae, which we are calling RNR2. We demonstrated that the gene is essential for mitotic viability and that its expression is induced by the DNAdamaging agent 4-nitroquinoline 1-oxide (4-NQO). We also investigated the damage inducibility of genes CDC8 and POLI and demonstrated that CDC8 is inducible whereas POLI is not. Our attention was originally drawn to the RNR2 protein due to a serendipitous cross-reaction with antibodies directed against the E. coli RecA protein.
(ribonucleotide reductase). Due to its central role in the control of DNA synthesis, ribonucleotide reductase has been extensively studied in a number of organisms. Among eucaryotes, the mammalian enzyme has been characterized in the greatest detail. It is composed of two nonidentical subunits, Ml and M2. The larger subunit, Ml, has been purified to homogeneity from calf thymus and is a dimer of molecular weight 170,000. It contains the binding sites for deoxynucleoside triphosphates which act as allosteric regulators of the enzymatic activity (36, 38). The smaller subunit, M2, is a dimer of molecular weight 88,000. It contains stoichiometric amounts of a nonheme iron center and a tyrosyl free radical which are essential for activity (38). These characteristics are typical of other eucaryotic and viral ribonucleotide reductases and are similar to those of the enzymes from Escherichia coli and phage T4 (30, 39). The coordination of events before and during DNA synthesis is critical for the proper replication and maintenance of chromosomes during mitosis. The extensive regulation of ribonucleotide reductase synthesis and activity reflects this required precision. The activity of the mammalian enzyme is allosterically inhibited by the final products of its pathway, the deoxynucleoside triphosphates (37). This ensures that only the proper concentration of precursors is produced and maintained. The activity is also cell cycle dependent, achieving maximal activity during S phase. This is accomplished by de novo synthesis and subsequent degradation of the M2 *
MATERIALS AND METHODS Affinity purification of antisera. Affinity-purified anti-RecA antibodies were prepared by a slight modification of the method of Smith and Fisher (31). A 100-,ug sample of RecA protein (gift of R. Bryant, Stanford University, Stanford, Calif.) was bound to a 2-cm2 piece of nitrocellulose for 30 min at room temperature, washed once with phosphatebuffered saline (PBS), and then incubated for 1 h in PBS plus
Corresponding author. 2783
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20% fetal calf serum to block the rest of the filter. The filter was washed three times with 20 ml of PBS and then incubated with 20 ml of rabbit anti-RecA serum (gift of I. R. Lehman) for 1 h at 4°C. The nitrocellulose was then washed five times with 20 ml of PBS plus 0.1% Nonidet P-40 at 4°C. Antibodies were eluted with two consecutive 30-s washes of 1 ml of 0.2 M glycine hydrochloride (pH 2.5), and the eluant was immediately neutralized by the addition of 0.5 volume of 1.0 M KPO4 (pH 9.0) + 5% fetal calf serum and then added to 20 ml of PBS plus 5% fetal calf serum. These antibodies were then diluted 1:100 into PBS plus 5% fetal calf serum and used directly for immunoblot analysis and library screening. Sera before purification gave several bands when used to probe both yeast and E. coli proteins, but yielded only one major cross-reactive protein with each after affinity purification. Immunoblot analysis. Yeast proteins were produced by taking 50 ml of logarithmically growing S. cerevisiae cells, washing them in 10 ml of 1.1 M sorbitol, and suspending them in 4 ml of 1.1 M sorbitol-50 mM KHPO4 (pH 6.8)-2 mg of Zymolyase per ml for 1 h at 30°C. Cells were washed three times with 10 ml of 1.1 M sorbitol, suspended in 2 ml of lysis buffer (40 mM Tris, pH 8, 1% sodium dodecyl sulfate, 50 mM dithiothreitol, and 50% urea [wt/vol]), and heated at 90°C for 5 min. Bacterial cells were isolated from logarithmically growing cultures and suspended directly in lysis buffer and lysed as above. In experiments analyzing UV induction, cells were subjected to 260-nm UV light (100 J/m2) and then shaken at 30°C for 1 h in rich medium before harvesting. Sodium dodecyl sulfate-10% polyacrylamide gels were run and electroblotted onto nitrocellulose (BA85, Schleicher and Schuell) essentially as described by Burnette (6). Each filter was incubated with 20 ml of a 1:100 dilution of affinitypurified anti-RecA antiserum for 1 h at 4°C. All subsequent washes and incubations were carried out at this temperature. Filters were washed four times in PBS and then incubated with 125I-protein A (Amersham) for 1 h, followed by four 15-min washes in PBS. Filters were air dried and exposed to film. Screening of a yeast genomic Agtll library. Affinity-purified rabbit anti-RecA antiserum (diluted 1:100) was used to screen a yeast genomic Agtll library (a gift from M. Snyder, Stanford University) as previously described (32); 125I1 protein A was used for detection. E. coli derivative strain BNN124, deleted for the recA gene, was used as a host for screening to avoid interference by endogenous RecA protein. DNA sequencing. The 4.6-kilobase (kb) BamHI genomic restriction fragment containing RNR2 was cloned into pEMBL19 (10), and recombinants with the insert in each orientation were selected. A series of nested deletions was created in these plasmids by the DNase I method of Anderson (1). Templates for sequencing were prepared by superinfection by fl (40). For those recombinants which gave poor yields of template by this method, the inserts were subcloned onto M13 mpl9. Sequencing was performed as described earlier (27). Mapping of cloned fragments to specific yeast chromosomes. Yeast chromosomes from S. cerevisiae YNN329 were prepared in low-melt agarose plugs and deproteinized as described by Schwartz and Cantor (28). The plugs were melted at 65°C, and the molten agar was loaded into a preparative slot (0.2 by 0.2 by 8 cm) in a 1% agarose gel. The orthogonalfield-alternation gel electrophoresis (8) was run by Marjorie Thomas at 300 V for 12 h at a 50-s switching frequency (7). The ethidium bromide-stained gel was treated with short-
MOL. CELL. BIOL.
wave UV light at 200 J/m2 for 1 min, the DNA was transferred to nitrocellulose (33), and the filter was cut into four pieces. Each piece was probed with a different probe. The YRp14 (ARSI CENIO) and YRp14 (ARSJ CENII) plasmids used as markers were obtained from P. Hieter (15). Enzymes, reactions, and media. Restriction enzymes, T4 DNA ligase, and calf intestine phosphatase were purchased from New England Biolabs or Boehringer-Mannheim and used under the conditions suggested by the supplier. Bacterial transformations were carried out as previously described (9). Medium components were from Difco or Sigma. Yeast media were as described by Sherman et al. (29) and Hieter et al. (15). DNA blot, RNA blot, and plaque hybridizations. DNA was labeled by the hexamer primer method of Feinberg and Vogelstein (13). Hybridizations for Southern blots were carried out as previously described (9). Plaque lifts and hybridizations were performed as described by Benton and Davis (4). The AEMBL 3A library of S. cerevisiae DNA used to obtain the genomic clones of RNR2 and the class 2 gene was a gift of M. Snyder, Stanford University. RNA was prepared from 4NQO-treated cells as described by Robinson et al. (25) and was a gift from C. Nicolet, Stanford University. RNA was resolved on formaldehyde-1% agarose gels as described by Maniatis et al. (22), and hybridizations were as described for the Southern analysis above. Transplason mutagenesis. The NNX 263 clone containing a lacZ-RNR2 fusion was mutagenized with mini-TnJO (TRPI Kan9 as described by Snyder et al. (32). Briefly, 108 logarithmically growing BNN123 cells were infected with NNX 263 at a multiplicity of infection of 2. They were incubated with shaking at 37°C for 2 h and then lysed with CHC13. This mutagenized stock was cleared of bacterial debris by centrifugation and used to infect BNN91, an hflA strain. Kanr lysogens of BNN91 were selected at 30°C, pooled, grown to mid-log phase (2 x 108 cells per ml), and shifted to 42°C for 2 h with shaking to thermally induce the lysogenic phage. These phage were then plated on strain BNN124 and immunoscreened. Phage which showed a loss of signal or reduced signal were chosen for analysis. Since the inserts into Xgtll were EcoRI fragments and the transplason does not contain an EcoRI site, we subcloned the EcoRI fragments from the two mutant phage, NNX 263-312, and 263-314, onto pUC19 to facilitate manipulation. These plasmids were then cleaved with EcoRI and used to transform the diploid S. cerevisiae strain YNN330 to Trp+. Yeast strains and transformations. The following strains of S. cerevisiae which are all derivatives of S-288C, were used: YNN329 (a ura3-52 ade2-101 lys2-801 Atrpl-901, Ahis3-200) and YNN330 (a/a ura3-521ura3-52 ade2-JOJIade2-101 Atrpl90IAtrpl-901 lys2-8011lys2-801 Ahis3-200/Ahis3-200). Yeast transformations were performed by selecting for Ura+ tranformants for pNN315, pNN316, and pNN317 and by selecting for Trp+ transformants for the transplacement reactions by using the lithium acetate procedure (16). All transplacements were verified by Southern hybridization analysis. Strain SK46a(pSR16) is described by Robinson et al. (25). Bacterial strains and plasmids used and constructed. Strain BNN124 was constructed by transducing the TnJO-linked A(srlR-recA) deletion of strain JC10289 (42) into strain LE392 by P1 transduction of the Tetr marker. Tetr transductants were checked for UV sensitivity, and a typical UV' colony was designated BNN124 and shown to lack RecA protein by immunoblotting. BNN123 is pNN314 [miniTnJO (TRP1 Kanr) in BNN114, all of which was described
RIBONUCLEOTIDE REDUCTASE GENE OF S. CEREVISIAE
VOL. 7, 1987
previously (32). pNN315, the RNR2 subclone onto YEp24 (5), was made by cloning the 4.6-kiobase (kb) BamHI fragment from a genomic clone of RNR2 in XEMBL 3A onto YEp24 cleaved with BamHI. pNN316, the class 2 subclone, was made by cloning the 5.2-kb EcoRI fragment of the XEMBL 3A clone of the class 2 gene into calf intestine alkaline phosphatase-treated, EcoRI-cleaved YEp24. pNN317 contains RNR2 URA3 CEN4 ARS1, and its construction will be described elsewhere. pEMBL19 (10) was obtained from W. Segraves. The centromere-containing plasmids, YRp14 (ARSJ CENIO) and YRp14 (ARS1 CENII), were obtained from P. Hieter (15). RESULTS Isolation of genes encoding immunoreactive proteins. Initially we were searching for a protein in S. cerevisiae which might serve as a recombinase. Using antibodies to the E. coli RecA protein as a probe, we performed an immunoblot analysis (Fig. 1) of S. cerevisiae proteins to identify proteins sharing antigenic determinants with the RecA protein. A strong cross-reaction with a protein of molecular weight 44,000 and a weaker reaction with several smaller proteins was found. We also detected a 1.5- to 2-fold increase in the immunosignal for the 44-kilodalton (kDa) protein upon UV irradiation of the yeast cells. We did not see a larger effect with other irradiation conditions, but Angulo et al. (2) identified the same protein with anti-RecA antibodies and were able to observe a fourfold increase after irradiation. Encouraged by the strong cross-reaction, we decided to isolate the gene encoding this protein. A genomic library of S. cerevisiae DNA fused to the lacZ gene of E. coli in Xgtll was screened immunologically with antibody to the E. coli RecA protein. To eliminate immunoreactive background due to endogenous RecA protein, we used E. coli BNN124, which has the recA gene deleted, as plating bacteria for the screening of this library. Five positive clones were identified and plaque purified from the 106 plaques screened on duplicate filters. Restriction endonuclease cleavage and Southern hybridization analysis (data not shown) showed these phage fell into two classes. Class 1 was composed of four members, E. coli UV
38k Da-
-
+
_
Yeast -
+
,__.
-
44 kDa
FIG. 1. Identification of a 44-kDa yeast protein that cross-reacts with anti-RecA antibodies. A 10-p.g sample of E. coli or S. cerevisiae (Yeast) protein from UV-induced or uninduced cells was subjected to electrophoresis in a 10%o sodium dodecyl sulfate-polyacrylamide gel, transferred to nitrocellulose, and then probed with affinitypurified polyclonal anti-RecA antiserum and 125I-protein A as described in Materials and Methods.
1
44 kDa -----
2
3
4
-
w
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-
FIG. 2. Identification of the gene encoding the 44-kDa protein by overproduction in S. cerevisiae. Genomic sequences for the two classes of genes were cloned onto the high-copy-number vector YEp24. Class 1 (RNR2), represented by pNN315, and class 2 genes, represented by pNN316, were introduced into S. cerevisiae YNN329 by lithium acetate transformation. Samples (10 ,ug) of protein from each of the strains YNN329(YEp24), YNN329(pNN315), and YNN329(pNN316) were subjected to electrophoresis in a 10o sodium dodecyl sulfate-polyacrylamide gel, transferred to nitrocellulose, and then probed with affinity-purified polyclonal anti-RecA antiserum as described in Materials and Methods. Two independent isolates of YNN329(pNN315) were used in this experiment. Lane 1, YNN329(YEp24); lane 2, YNN329 (pNN316); lanes 3 and 4, independent isolates of YNN329 (pNN315).
two of which appeared to be identical. Class 2 was represented by only one clone and had a much weaker immunoreactivity. To isolate the full-length genes represented by these classes, we obtained genomic clones representing each class from a XEMBL 3A genomic library of yeast DNA. Identification of the gene encoding the 44-kDa immunoreactive protein by overproduction in S. cerevisiae. To identify the proteins encoded by these genomic DNA clones, we attempted to express the genes at high levels in S. cerevisiae. For class 1, a 4.6-kb BamHI fragment was subcloned onto the multicopy vector YEp24 to make pNN315. This fragment was chosen because it had at least 2 kb of DNA on both sides of the fusion break point in NNX 263. For class 2, a 5.2-kb EcoRI fragment was subcloned onto YEp24 to make pNN316. These plasmids were transformed into S. cerevisiae YNN329 by selection for Ura prototrophy. Immunoblot analysis was performed on protein from these transformants (Fig. 2). Only clones of class 1 DNA, when present on YEp24, were able to overproduce the 44-kDa immunoreactive protein. The class 2 gene gave the same pattern as the strain containing YEp24 alone. Therefore, we conclude that the class 1 gene encodes the 44-kDa protein. Sequence of the gene encoding the 44-kDa protein reveals homology to ribonucleotide reductase. The nucleotide sequence of the gene encoding the 44-kDa protein and its deduced amino acid sequence are shown in Fig. 3. There is an open reading frame of 399 amino acids, enough to produce a protein of 46 kDa. The deduced amino acid sequence was compared with the sequence of the RecA protein. The only significant homology occurred at the last four amino acids of the two proteins (Fig. 4). Although this seems rather small for an epitope, information presented below indicates that it is likely to be solely responsible for the cross-reaction. A search of the National Institutes of Health protein data base for homologous proteins revealed
ELLEDGE AND DAVIS
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CACCACGCGC GATCGCATGG CAACGAGGTC GCACACGCCC CACACCCAGA CCTCCCTGCG AGCGGGCATG GGTACAATGT CCCCGTTGCC ACAGAGACCA CTTCGTAGCA CAGCGCAGCA
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GCTAGCTGGT TGTTGCTGCT GACAAAAGAA AATTTTTCTT AGCAAAAGGA GGGGAAGCAC GGGCAGATAG CACCGTACCA TACCCTTGGA AACTCGAAAT GAACGAAGCA GGAAATGAGA
250 260 270 280 290 300 310 320 330 340 350 360 GAATGAGAGT TTTGTAGGTA TATATAGCGG TAGTGTTTGC GCGTTACCAT CATCTTCTGG ATCTATCTAT TGTTCTTTTC CTCATCACTT TCCCCTTTTT CGCTCTTCTT CTTGTCTTTT
370 380 390 400 410 420 430 440 462 ATTTCTTTCT TTTTTTTAAT TGTTCCCTCG AATGGCTATC TACCAAAGAA TACAAACTTA ATACACGTAT TTATTTGTCC AATTACC ATG CCT AAA GAG ACC CCT TCC AAA GCT MET Pro Lys Glu Thr Pro S-r Lys Ala
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GCT GCC GAT GCA TTG TCC GAC TTG GAA ATC AAA GAT TCC AAG TCC AAC CTT AAC AAG GAA TTG GAG ACA TTG AGA GAG GMA AAC AGA GTA AAG TCA GAC Ala Ala Asp Ala Lou Ser Asp Lou Glu Ile Lys Asp Ser Lys Ser Asn Leu Asn Lys Glu Leu Glu Thr Leu Arg Glu Glu Asn Arg Val Lys Ser Asp
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ATG CTT AAG GAG AAA TTG AGC AAG GAC GCT GAA AAT CAC AAG GCT TAC TTG AAA TCT CAT CAA GTT CAC CGT CAC AAA CTT AAG GAA ATG GAA AAG GAG MET Leu Lys Glu Lys Leu Ser Lys Asp Ala Glu Asn His Lys Ala Tyr Leu Lys Ser His Gln Val His Arg His Lys Lou Lys Glu MET Glu Lys Glu
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GAA CCT TTG TTG AAT GAA GAC AAG GAG AGA ACT GTT CTT TTC CCT ATC AAG TAC CAT GAA ATC TGG CAA GCC TAC GMA GCT TCT TTC TGG ACC GCT GAA Glu Pro Leu Leu Asn Glu Asp Lys Glu Arg Thr Val Leu Phe Pro Ile Lys Tyr His Glu Ile Trp Gln Ala Tyr Lys Arg Ala Glu Ala Ser Phe Trp
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ACC GCT GAA GAA ATT GAT TTG TCT AAG GAT ATC CAT GAC TGG AAC AAC AGA ATG AAC GAA AAC GAG AGA TTT TTC ATT TCC AGA GTT CTT GCC TTT TTC Thr Ala Glu Glu Ile Asp Leu Ser Lys Asp Ile His Asp Trp Asn Asn Arg MET Asn Glu Asn Glu Arg Phe Phe Ile Ser Arg Val Leu Ala Phe Phe
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GCC GCT TCT GAC GGT ATT GTT AAT GAA AAC TTG GTT GAA AAC TTC TCC ACC GAA GTC CAA ATT CCA GAG GCA AAG AGT TTC TAC GGT TTC CAA ATC ATG Ala Ala Ser Asp Gly Ile Val Asn Glu Asn Leu Val Glu Asn Phe Ser Thr Glu Val Gln Ile Pro Glu Ala Lys Ser Phe Tyr Gly Phe Gln Ile MET
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ATT GAA AAT ATT CAC TCT GAA ACT TAC TCC TTG TTG ATC GAT ACT TAC ATC AAG GAC CCT AAA GAA AGT GAA TTC TTG TTC AAT GCC ATT CAC ACC ATC Ile Glu Asn Ile His Ser Glu Thr Tyr Ser Leu Leu Ile Asp Thr Tyr Ile Lys Asp Pro Lys Glu Ser Glu Phe Leu Phe Asn Ala Ile His Thr Ile
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CCA GAA ATC GGT GAG AAG GCC GAA TGG GCT TTA AGA TGG ATT CAA GAC GCT GAC GCC TTG TTT GGT GAA AGA CTA GTT GCC TTT GCC TCC ATT GMA GGT Pro Glu Ile Gly Glu Lys Ala Glu Trp Ala Leu Arg Trp Ile Gln Asp Ala Asp Ala Lou Phe Gly Glu Arg Leu Val Ala Pho Ala Ser Ilo Glu Gly
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GTC TTT TTC TCC GGT TCC TTT GCC TCC ATT TTC TGG TTG AAA AAG AGA GGT ATG ATG CCC GGT TTA ACC TTT TCC AAC GAA TTG ATC TGT AGA GAC GAA Val Phe Phe Ser Gly Ser Phe Ala Ser Ile Phe Trp Leu Lys Lys Arg Gly MET MET Pro Gly Leu Thr Phe Ser Asn Glu Lou Ile Cys Arq Asp Glu
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GGT TTG CAC ACC GAC TTT GCA TGC TTG TTG TTC GCC CAT TTG AAG AAC AAA CCA GAC CCA GCC ATT GTT GAA AAA ATT GTC ACC GAG GCT GTG GAA ATT Gly Leu His Thr Asp Phe Ala Cys Leu Leu Phe Ala His Leu Lys Asn Lys Pro Asp Pro Ala I1e Val Glu Lys Ile Val Thr Glu Ala Val Glu Ile
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GAA CAA AGA TAC TTC TTG GAC GCC TTA CCA GTT GCT TTG CTA GGT ATG AAC GCT GAC TTA ATG AAC CAA TAC GTT GAG TTC GTC GCC GAC AGA CTG TTG Glu Gln Arq Tyr Ph. Leu Asp Ala Leu Pro Val Ala Leu Leu Gly MET Asn Ala Asp Leu MET Asn Gln Tyr Val Glu Pho Val Ala Asp Arg Leu Leu
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GTT GCT TTC GGT AAC AAG AAA TAC TAC AAG GTC GAA AAC CCC TTC GAT TTC ATG GAA AAC ATC TCC TTG GCC GGT AAG ACC AAC TTC TTC GAA AAG AGA Val Ala Phe Gly Asn Lys Lys Tyr Tyr Lys Val Glu Asn Pro Phe Asp Phe MET Glu Asn Ile Ser Lou Ala Gly Lys Thr Asn Phe Phe Glu Lys Arg
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GTT TCT GAC TAC CAA AAG GCT GGT GTT ATG TCC AAG TCG ACT AAG CAA GAA GCC GGT GCT TTC ACC TTC AAC GAA GAC TTT Val Ser Asp Tyr Gln Lys Ala Gly Val MET Ser Lys Ser Thr Lys Gln Glu Ala Gly Ala Phe Thr Phe Asn Glu Asp Phe
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TAAGAGTATC TCTCTATATA
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TTTCTTTTTA CGCAGTCTCT TCAATCTCTT TATGTGGGCT TTCGCACCTC TTATTTATAT ATATATATAT ACACTGAATC ATATAAACTT TTTTTTATAT TGAATCTAAT ATATTATCAA
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ACGGAAACTT CGGCTGAATT TCATACGTAT ATTGATTAAA GTGGAAAGGG CATCGGAAAA GTAAGAAAAG CTTAAAAAAA
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AAAAACCGTA TACAATACAT ACATATGTAT ATGAATATAA CCGTGGTACG ATGTGTCTTA AAAAATAGTT GAACGAACTA CTCGGACATA CCCCTACATC ACTTAAGGAT GTCTACTTTC
2034 2044 2054 2064 2074 2084 2094 ATAAGTGGTC CCATCTGCGG TACAGACAAC TGCCCCTCAC GGCTTGGCTA TCATTGATGG GAGAAGAACC
FIG. 3. Nucleotide sequence of the RNR2 gene encoding the small subunit of ribonucleotide reductase and the deduced amino acid sequence. Nucleotides are numbered from a C, which lies 10 nucleotides before the unique PvuI site shown in Fig. 7, and proceed for 2,094 nucleotides.
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one protein with extensive homology, the small subunit of ribonucleotide reductase encoded by herpes simplex virus type 2. Figure 5 compares the deduced amino acid sequence of the S. cerevisiae gene with those of the corresponding genes from mouse, clam, herpes simplex virus type 2, Epstein-Barr virus, E. coli, and bacteriophage T4. Gaps
have been introduced to maximize the homology between them. The greatest amount of homology exists between the mouse and clam sequences (82%). It is probable that the clam protein is actually larger than reported (34) because translation of the sequences directly preceding the suggested initiation codon would extend the amino acid homology to both the mouse and yeast genes. The yeast protein has two possible initiation codons at positions 1 and 43. However, homology between the yeast protein and the mouse protein within those first 43 amino acids suggests that the ATG at position 1 is the authentic initiation codon. The extent of homology between the yeast protein and the others shown in Fig. 5 is presented in Table 1. These percentages are for exact matches and roughly follow what one would predict from phylogenetic lineages. Of the 399 amino acids present in ribonucleotide reductase, only 16 appear to be totally conserved in the seven proteins analyzed here. Twelve residues are conserved in six of the seven species examined. Among the 16 totally conserved residues are the tyrosine at residue 194, which carries the free radical involved in the catalytic mechanism, and the histidine at residue 190, which is coordinated to the iron center also required for catalytic activity. The differences among these seven distantly related proteins constitute a saturation mutagenesis conducted by the process of evolution. Hence, it is likely that mutagenesis of any of these 16 conserved amino acids would result in a severe loss of function. Mapping of RNR2 to chromosome X. Chromosomes from strain YNN329 were prepared and separated on a 1% agarose gel by using an orthogonal-field-alternation gel electrophoresis apparatus. The chromosomes were blotted onto nitrocellulose and analyzed by Southern hybridization (Fig. 6). RNR2 DNA hybridizes to chromosome X, and class 2 DNA hybridizes to chromosome XI. The identification of chromosomes X and XI was confirmed by using plasmids containing centromeric DNA from chromosomes X and XI as a probe. Genetic mapping data discussed below indicate that RNR2 is tightly linked to the centromere. Identification of the epitope coding region and disruption of RNR2 by using transplason mutagenesis. We used the technique of transplason mutagenesis to identify which regions of the Xgtll clones encode the cross-reacting antigen(s). This technique allows the rapid identification of epitope coding regions by transposon mutagenesis, as well as the susequent mutagenesis of the chromosomal copy of the gene by transplacement. Transpositions of mini-TnJO (TRPJ Kan) onto NNX 263 were selected for as described (32). NNX 263, whose insert is outlined in Fig. 7, was chosen because it 396
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-Asn-Glu-Asp-Phe-COOH 350
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-Asn-Glu-Asp-Phe-COOH RecA FIG. 4. Homology between the E. coli RecA protein and yeast ribonucleotide reductase. The sequences shown here represent the best match as determined by a computer comparison of the two protein sequences. These two sequences represent the carboxy terminus of each protein.
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TABLE 1. Exact amino acid identity shared between the small subunit of ribonucleotide reductase of S. cerevisiae and other organismsa Organism % Identity Clam ........................................... 67 Mouse ........................................... 60 ................... 24 Epstein-Barr virus .............. Herpes simplex virus type 2 ......................... 23 E. coli ...................................... 17 T4 ................................................ 15 a Percentages were calculated by taking the total number of exact amino acid matches between the yeast protein and the various homologs, dividing by the number of amino acids in the shorter of the two proteins, and multiplying by 100.
encoded a lacZ-RNR2 fusion protein and gave a very strong signal upon immunoscreening. Approximately 1,000 independent transposition events selected as Kanr lysogens in BNN91 were pooled, thermally induced, and plaqued on BNN124, the recA deletion strain. Approximately 1,000 plaques were immunoscreened. Phage which showed a loss of signal or reduced signal were chosen for further analysis. In this experiment, all phage which appeared to have a reduced signal did not retain that phenotype after further purification. Twelve phage which demonstrated a loss of signal were chosen for restriction analysis. Of these, 10 had inserted into the lacZ gene and 2 had transposed into the yeast DNA. The positions of the transposition events relative to the coding regions and restriction map of the gene are illustrated in Fig. 7. Since the transplason carries the TRPJ gene, it is a simple matter to transplace the mutations thus created onto the chromosome. Mutagenized inserts from NNX 263-312 and 263-314 were used to transform the diploid S. cerevisiae strain YNN330 to Trp+ as described in Materials and Methods. Three independent Trp+ transformants were purified and placed on sporulation medium. Of 35 tetrads analyzed for the 312 disruption, no tetrads with three or four viable spores were observed (Table 2). Fifteen tetrads had two viable spores and 10 tetrads had one viable spore, and all viable spores were Trp auxotrophs. This demonstrates that the 312 insertion disrupts an essential function. Introduction of a centromere containing a plasmid carrying RNR2 and URA3, pNN317, into the heterozygous diploid rescued the viability of Trp+ spores. All Trp+ spores were Ura+, suggesting that the presence of the plasmid was essential for viability of those spores. The presence of URA3 on a centromere-containing plasmid gave us the opportunity to analyze centromere linkage. Only 1 of 14 informative tetrads gave a second-division segregation (SDS) event between the chromosomal RNR2 locus and URA3. The MAT locus, which shows a weaker centromere linkage of 20 centimorgans, gave only 3 SDS events out of 21 informative tetrads. Using the formula for centromere linkage of Sherman et al. (31), (T)AB = (SDS)A + (SDS)B -3/2 (SDS)A(SDS)B, where numbers in parentheses represent frequencies, we obtain a value of TAB = 0.2 and a centromere linkage value of 10 centimorgans. Although the number of informative tetrads is low, the centromere linkage is strengthened through the demonstration of tight linkage to two different centromere-linked markers. The value of 10 centimorgans of linkage is likely to be an overestimate because of the weak linkage of the MAT locus. The 314 insertion was more complicated. Although it interrupted the gene very near its 3' end, within 100 base pairs, we were unable to isolate simple insertions that were
ELLEDGE AND DAVIS
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