The Meiosis-specific Protein Kinase Ime2 Directs Phosphorylation of Replication Protein A
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m306943200
ISSN1083-351X
AutoresDawn M. Clifford, Suzanne M. Marinco, George S. Brush,
Tópico(s)Microtubule and mitosis dynamics
ResumoIn Saccharomyces cerevisiae, the cellular single-stranded DNA-binding protein replication protein A (RPA) becomes phosphorylated during meiosis in two discrete reactions. The primary reaction is first observed shortly after cells enter the meiotic program and leads to phosphorylation of nearly all the detectable RPA. The secondary reaction, which requires the ATM/ATR homologue Mec1, is induced upon initiation of recombination and only modifies a fraction of the total RPA. We now report that correct timing of both RPA phosphorylation reactions requires Ime2, a meiosis-specific protein kinase that is critical for proper initiation of meiotic progression. Expression of Ime2 in vegetative cells leads to an unscheduled RPA phosphorylation reaction that does not require other tested meiosis-specific kinases and is distinct from the RPA phosphorylation reaction that normally occurs during mitotic growth. In addition, immunoprecipitated Ime2 catalyzes phosphorylation of purified RPA. Our data strongly suggest that Ime2 is an RPA kinase in vivo. We propose that Ime2 directly catalyzes RPA phosphorylation in the primary reaction and indirectly promotes the Mec1-dependent secondary reaction by advancing cells through meiotic progression. Our studies have identified a novel meiosis-specific reaction that targets a key protein required for DNA replication, repair, and recombination. This pathway could be important in differentiating mitotic and meiotic DNA metabolism. In Saccharomyces cerevisiae, the cellular single-stranded DNA-binding protein replication protein A (RPA) becomes phosphorylated during meiosis in two discrete reactions. The primary reaction is first observed shortly after cells enter the meiotic program and leads to phosphorylation of nearly all the detectable RPA. The secondary reaction, which requires the ATM/ATR homologue Mec1, is induced upon initiation of recombination and only modifies a fraction of the total RPA. We now report that correct timing of both RPA phosphorylation reactions requires Ime2, a meiosis-specific protein kinase that is critical for proper initiation of meiotic progression. Expression of Ime2 in vegetative cells leads to an unscheduled RPA phosphorylation reaction that does not require other tested meiosis-specific kinases and is distinct from the RPA phosphorylation reaction that normally occurs during mitotic growth. In addition, immunoprecipitated Ime2 catalyzes phosphorylation of purified RPA. Our data strongly suggest that Ime2 is an RPA kinase in vivo. We propose that Ime2 directly catalyzes RPA phosphorylation in the primary reaction and indirectly promotes the Mec1-dependent secondary reaction by advancing cells through meiotic progression. Our studies have identified a novel meiosis-specific reaction that targets a key protein required for DNA replication, repair, and recombination. This pathway could be important in differentiating mitotic and meiotic DNA metabolism. Meiosis is a specialized process in which a single diploid cell undergoes one round of DNA replication followed by two consecutive rounds of nuclear division to generate four haploid progeny. For most species examined, the first nuclear division is directly preceded by a programmed recombination phase that is required for proper chromosome segregation and also serves to enhance genetic variability. In multicellular eukaryotes, this remarkable capacity to both rearrange chromosomal content and halve the chromosome number is restricted to a subset of cells destined to form gametes. However, single-celled eukaryotes such as the budding yeast Saccharomyces cerevisiae can also undergo meiosis, leading to the production of haploid spores upon starvation. In either case, the reduction in ploidy resulting from meiosis is a prerequisite to zygote formation, whereupon the cellular DNA content is returned to a diploid state. Studies in a variety of systems have revealed that regulation of chromosome dynamics and maintenance of genomic integrity during meiosis require the induction of numerous proteins upon commitment of cells to meiotic differentiation. Among these are the meiosis-specific protein kinases that initiate and/or propagate critical meiotic signaling pathways. In S. cerevisiae, the protein kinase Ime2 is an important regulator of meiotic initiation (1Smith H.E. Mitchell A.P. Mol. Cell. Biol. 1989; 9: 2142-2152Crossref PubMed Scopus (150) Google Scholar, 2Mitchell A.P. Driscoll S.E. Smith H.E. Mol. Cell. Biol. 1990; 10: 2104-2110Crossref PubMed Scopus (108) Google Scholar, 3Yoshida M. Kawaguchi H. Sakata Y. Kominami K. Hirano M. Shima H. Akada R. Yamashita I. Mol. Gen. Genet. 1990; 221: 176-186Crossref PubMed Scopus (96) Google Scholar, 4Kominami K. Sakata Y. Sakai M. Yamashita I. Biosci. Biotechnol. Biochem. 1993; 57: 1731-1735Crossref PubMed Scopus (34) Google Scholar, 5Foiani M. Nadjar-Boger E. Capone R. Sagee S. Hashimshoni T. Kassir Y. Mol. Gen. Genet. 1996; 253: 278-288Crossref PubMed Scopus (63) Google Scholar). Ime2 is induced early in meiosis and is required for maximal induction of early, middle, and late meiosis-specific genes. In the absence of Ime2 function, cells do not progress normally into "pre-meiotic" DNA replication and are deficient in completing downstream events in the sporulation process. Recent studies have demonstrated that Ime2 isolated by immunoprecipitation catalyzes phosphorylation of Ndt80 (6Sopko R. Raithatha S. Stuart D. Mol. Cell. Biol. 2002; 22: 7024-7040Crossref PubMed Scopus (56) Google Scholar, 7Benjamin K.R. Zhang C. Shokat K.M. Herskowitz I. Genes Dev. 2003; 17: 1524-1539Crossref PubMed Scopus (225) Google Scholar), a meiosis-specific transcription factor that is required for up-regulation of middle sporulation genes (8Xu L. Ajimura M. Padmore R. Klein C. Kleckner N. Mol. Cell. Biol. 1995; 15: 6572-6581Crossref PubMed Scopus (261) Google Scholar, 9Hepworth S.R. Friesen H. Segall J. Mol. Cell. Biol. 1998; 18: 5750-5761Crossref PubMed Google Scholar, 10Chu S. Herskowitz I. Mol. Cell. 1998; 1: 685-696Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Generation of phosphorylated Ndt80 correlates with maximal Ndt80 activation, suggesting that Ime2 enhances Ndt80 activity through phosphorylation (6Sopko R. Raithatha S. Stuart D. Mol. Cell. Biol. 2002; 22: 7024-7040Crossref PubMed Scopus (56) Google Scholar, 7Benjamin K.R. Zhang C. Shokat K.M. Herskowitz I. Genes Dev. 2003; 17: 1524-1539Crossref PubMed Scopus (225) Google Scholar). Another study has shown that Ime2 immunoprecipitates catalyze phosphorylation of Ime1 (11Guttmann-Raviv N. Martin S. Kassir Y. Mol. Cell. Biol. 2002; 22: 2047-2056Crossref PubMed Scopus (62) Google Scholar), an early meiosis-specific transcription factor (12Kassir Y. Granot D. Simchen G. Cell. 1988; 52: 853-862Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 13Smith H.E. Driscoll S.E. Sia R.A. Yuan H.E. Mitchell A.P. Genetics. 1993; 133: 775-784Crossref PubMed Google Scholar). In this case, Ime2-mediated phosphorylation is thought to negatively regulate Ime1 by promoting its degradation (11Guttmann-Raviv N. Martin S. Kassir Y. Mol. Cell. Biol. 2002; 22: 2047-2056Crossref PubMed Scopus (62) Google Scholar). There is also evidence that Ime2 regulates proteins that are more directly associated with controlling meiotic progression, such as the B-type cyclin inhibitor Sic1 (14Dirick L. Goetsch L. Ammerer G. Byers B. Science. 1998; 281: 1854-1857Crossref PubMed Scopus (114) Google Scholar) and the anaphase-promoting complex ubiquitin ligase activator Cdh1 (15Bolte M. Steigemann P. Braus G.H. Irniger S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4385-4390Crossref PubMed Scopus (40) Google Scholar). Further studies will be required to determine whether Ime2 catalyzes phosphorylation of these proteins. Given that Ime2 is required for proper pre-meiotic DNA replication, other potential targets of Ime2 protein kinase activity include proteins that are involved in the initiation of DNA synthesis. One candidate is replication protein A (RPA), 1The abbreviations used are: RPAreplication protein AssDNAsingle-stranded DNAHahemagglutinin epitopeIme2-His/Hahistidine and hemagglutinin epitope-tagged version of Ime2Ime2kd-His/Hakinase-dead version of Ime2-His/HaSPMsporulation mediumDTTdithiothreitol. an abundant single-stranded DNA (ssDNA)-binding protein that is required for DNA replication, repair, and recombination (for review see Ref. 16Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1208) Google Scholar). RPA is an evolutionarily conserved heterotrimeric complex, and each subunit is essential for viability in yeast (17Heyer W.D. Rao M.R.S. Erdile L.F. Kelly T.J. Kolodner R.D. EMBO J. 1990; 9: 2321-2329Crossref PubMed Scopus (159) Google Scholar, 18Brill S.J. Stillman B. Genes Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (199) Google Scholar). A fundamental role of RPA is to stabilize the ssDNA that is generated during DNA transactions. RPA also interacts with several other proteins required for DNA replication, repair, and recombination. Consequently, RPA is likely to play an important regulatory role in DNA metabolism. Consistent with this hypothesis, RPA is phosphorylated periodically during the mitotic cell cycle and also in response to genotoxic stress in both human and yeast cells (19Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (205) Google Scholar, 20Din S. Brill S.J. Fairman M.P. Stillman B. Genes Dev. 1990; 4: 968-977Crossref PubMed Scopus (246) Google Scholar, 21Liu V.F. Weaver D.T. Mol. Cell. Biol. 1993; 13: 7222-7231Crossref PubMed Scopus (189) Google Scholar, 22Carty M.P. Zernik-Kobak M. McGrath S. Dixon K. EMBO J. 1994; 13: 2114-2123Crossref PubMed Scopus (169) Google Scholar, 23Brush G.S. Morrow D.M. Hieter P. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15075-15080Crossref PubMed Scopus (166) Google Scholar, 24Brush G.S. Kelly T.J. Nucleic Acids Res. 2000; 28: 3725-3732Crossref PubMed Scopus (80) Google Scholar). replication protein A single-stranded DNA hemagglutinin epitope histidine and hemagglutinin epitope-tagged version of Ime2 kinase-dead version of Ime2-His/Ha sporulation medium dithiothreitol. Our recent studies have demonstrated that yeast RPA also becomes phosphorylated in at least two separate reactions during meiotic progression (25Brush G.S. Clifford D.M. Marinco S.M. Bartrand A.J. Nucleic Acids Res. 2001; 29: 4808-4817Crossref PubMed Scopus (23) Google Scholar). The primary RPA phosphorylation reaction first occurs soon after cells enter meiosis and would appear to coincide temporally with Ime2 induction. The secondary reaction occurs upon initiation of meiotic recombination and requires the protein kinase Mec1, an important regulator of DNA metabolic checkpoints (26Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-665Crossref PubMed Scopus (678) Google Scholar, 27Paulovich A.G. Hartwell L.H. Cell. 1995; 82: 841-847Abstract Full Text PDF PubMed Scopus (528) Google Scholar, 28Siede W. Allen J.B. Elledge S.J. Friedberg E.C. J. Bacteriol. 1996; 178: 5841-5843Crossref PubMed Google Scholar, 29Lydall D. Nikolsky Y. Bishop D.K. Weinert T. Nature. 1996; 383: 840-843Crossref PubMed Scopus (55) Google Scholar, 30Stuart D. Wittenberg C. Genes Dev. 1998; 12: 2698-2710Crossref PubMed Scopus (134) Google Scholar) that is also required for RPA phosphorylation events in vegetative cells (23Brush G.S. Morrow D.M. Hieter P. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15075-15080Crossref PubMed Scopus (166) Google Scholar, 24Brush G.S. Kelly T.J. Nucleic Acids Res. 2000; 28: 3725-3732Crossref PubMed Scopus (80) Google Scholar). Mec1 is homologous to the human protein kinase ATM, which is mutated in ataxia-telangiectasia (31Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanjali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jaspers N.G.J. Taylor A.M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.S. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2396) Google Scholar), and is even more homologous to the human "ATM- and Rad3-related" protein kinase ATR (for review see Ref. 32Rouse J. Jackson S.P. Science. 2002; 297: 547-551Crossref PubMed Scopus (578) Google Scholar). Despite the participation of Mec1, our data have suggested that the secondary RPA phosphorylation reaction is not involved in meiotic Mec1-dependent checkpoint delay processes. Instead, it is likely that Mec1-dependent RPA phosphorylation functions to regulate recombination itself. The possibility that phosphorylation modulates RPA function in DNA metabolism is supported by earlier studies employing the cell-free SV40 DNA replication system. These experiments have suggested that certain forms of phosphorylated human RPA are inefficient in supporting DNA replication and might preferentially function in DNA repair (22Carty M.P. Zernik-Kobak M. McGrath S. Dixon K. EMBO J. 1994; 13: 2114-2123Crossref PubMed Scopus (169) Google Scholar, 33Park J.S. Park S.J. Peng X. Wang M. Yu M.A. Lee S.H. J. Biol. Chem. 1999; 274: 32520-32527Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 34Liu J.S. Kuo S.R. McHugh M.M. Beerman T.A. Melendy T. J. Biol. Chem. 2000; 275: 1391-1397Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In order to further understand the function of RPA phosphorylation, we have investigated the mechanism underlying the sequential RPA phosphorylation reactions that occur during meiotic progression. We now demonstrate that proper timing of both primary and secondary meiotic RPA phosphorylation requires Ime2. Our genetic and biochemical evidence suggests that the initial meiosis-specific reaction is directly catalyzed by Ime2. The secondary Mec1-dependent reaction is indirectly activated by the Ime2-dependent promotion of meiotic progression. Nearly all of the detectable RPA becomes phosphorylated in the primary Ime2-catalyzed reaction, suggesting that this novel meiotic pathway plays a significant role in directing the meiosis-specific DNA metabolic functions of RPA. Strains—The following yeast strains derived from SK-1 (35Kane S.M. Roth R. J. Bacteriol. 1974; 118: 8-14Crossref PubMed Google Scholar) cells were used in this study: DSY1089 (wild type) (30Stuart D. Wittenberg C. Genes Dev. 1998; 12: 2698-2710Crossref PubMed Scopus (134) Google Scholar), MATa/α ho::LYS2/" lys2/" leu2::hisG/" trp1::hisG/" ura3/" arg4-BglII/arg4-NspI his4X/his4B; YGB221 (ime2Δ), MATa/α ho::LYS2/" lys2/" leu2::hisG/" trp1:: hisG/" ura3/" arg4-BglII/arg4-NspI his4X/his4B ime2::TRP1/"; YGB283 (sic1Δ), MATa/α ho::LYS2/" lys2/" leu2::hisG/" trp1::hisG/" ura3/" arg4/arg4-NspI his4/his4X sic1::kanMX4/"; YGB286 (ime2Δ sic1Δ), MATa/α ho::LYS2/" lys2/leu2::hisG/" trp1::hisG/" ura3/" arg4/" his4/" ime2::TRP1/" sic1::kanMX4/". A PCR-based strategy (36Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1137) Google Scholar) was used to replace a 2-kb fragment containing the entire open reading frame of IME2 with TRP1 in a haploid strain. This mutation was then transmitted to other strains by standard mating and sporulation protocols. A similar method was used to replace a 1.1-kb genomic region containing the SIC1 open reading frame with a 1.6-kb kanMX4 fragment. The following yeast strains containing a W303 (37Thomas B.J. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1388) Google Scholar) genetic background were also used: W303-1A (wild type), MATaade2-1 can1-100 his3-11,115 leu2-3,112 trp1-1 ura3-1; YGB23 (rfa2-S122D), MATaade2-1 can1-100 his3-11,115 leu2-3,112 trp1-1 ura3-1 rfa2-S122D; YGB138 (wild type), MATa/α ade2-1/" can1-100/" his3-11,115/" leu2-3,112/" trp1-1/" ura3-1/"; YGB140 (rfa2-S122A), MATa/α ade2-1/" can1-100/" his3-11,115/" leu2-3,112/" trp1-1/" ura3-1/" rfa2-S122A/". Mutant alleles of RFA2 encoding Rfa2 with serine-to-aspartate (S122D) or serine-to-alanine (S122A) substitutions at residue 122 were generated in progenitor haploids by "pop-in/pop-out replacement" (38Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1127) Google Scholar). All mutations generated in our laboratory were confirmed by PCR and Southern blot analysis. Analysis of ectopic Ime2 expression included ime2, mek1, rim15, smk1, and sps1 deletion mutants from the homozygous diploid deletion set (Invitrogen). Ime2 Plasmids—PCR-based cloning methods were employed to generate plasmids expressing C-terminally tagged versions of Ime2. A sequence encoding six histidine repeats followed by three hemagglutinin epitope (Ha) repeats was inserted at the NotI site of pRS426 (39Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1455) Google Scholar) to generate pRS426-His/Ha. Oligonucleotide primers designed to engineer an XmaI site upstream of the IME2 promoter and a NotI site immediately prior to the IME2 stop codon were used to amplify the entire IME2 gene from pAM405 (1Smith H.E. Mitchell A.P. Mol. Cell. Biol. 1989; 9: 2142-2152Crossref PubMed Scopus (150) Google Scholar), generously provided by Craig Giroux (Wayne State University). The PCR fragment was inserted directly into pGEM-T (Promega) by "TA" cloning. DNA sequencing revealed two PCR-generated mutations that were corrected by replacement with a restriction fragment from pAM405. IME2 was then excised from the pGEM-T clone with XmaI and NotI and inserted between the XmaI and NotI sites of pRS426-His/Ha. The resulting plasmid, pDC005, encodes a C-terminally tagged version of Ime2 designated Ime2-His/Ha. The plasmid pDC006, which encodes a kinase-dead mutant of Ime2 (Ime2kd-His/Ha) resulting from a lysine-to-alanine mutation at residue 97 (11Guttmann-Raviv N. Martin S. Kassir Y. Mol. Cell. Biol. 2002; 22: 2047-2056Crossref PubMed Scopus (62) Google Scholar), was generated from pDC005 by site-directed mutagenesis (QuikChange, Stratagene). Yeast cells were transformed with plasmids using a lithium acetate-based procedure (40Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Media, Growth, and Sporulation—Complex medium containing glucose (YPD) or glycerol (YPG) and synthetic complete medium lacking uracil were prepared as described (41Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 145-149Google Scholar). Sporulation medium (SPM) consisted of 0.3% potassium acetate and 0.02% raffinose supplemented with leucine at 0.0025% and arginine, histidine, tryptophan, and uracil each at 0.0005%. All incubations were carried out at 30 °C. SK-1 cells were synchronously sporulated as described (25Brush G.S. Clifford D.M. Marinco S.M. Bartrand A.J. Nucleic Acids Res. 2001; 29: 4808-4817Crossref PubMed Scopus (23) Google Scholar). Briefly, single colonies were selected following growth on YPG and incubated in YPD overnight. The overnight cultures were used to inoculate 200 ml of YPA (1% yeast extract, 2% bactopeptone, 2% potassium acetate) at an A600 of 0.2 for pre-sporulation growth. After 12 h of incubation, pre-sporulation cells were washed once with 20 ml of SPM, resuspended in 200 ml of SPM, and further incubated for the indicated times. Western Blot Analysis—To prepare denatured extracts, cells that had been washed with water and stored at -80 °C were resuspended in sample buffer (62.5 mm Tris, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromphenol blue) and subjected to three cycles of freezing (dry ice for 2 min) followed by heating (95 °C for 2 min). The suspensions were then centrifuged at 18,000 × g for 5 min at room temperature to pellet cell debris, and aliquots of the supernatants were further analyzed as described below. Immunoprecipitated and purified proteins (see below) were dissolved in sample buffer, heated at 95 °C for 10 min, and centrifuged at 18,000 × g for 30 s. To allow for reproducible detection of immunoprecipitated Ime2 by Western blot analysis, 20 μg of carrier protein (yeast crude extract devoid of Ime2-His/Ha) was added to the Ime2 immunoprecipitates. Samples to be analyzed for Rfa2 phosphorylation were subjected to electrophoresis through a 12% low cross-linking SDS-polyacrylamide gel (150:1 w/w acrylamide/bisacrylamide). For analyses not requiring such resolution, 10% SDS-polyacrylamide gels (37.5:1 w/w acrylamide/bisacrylamide) were employed. Separated proteins were transferred to nitrocellulose (0.2-μm pore size, Schleicher & Schuell) in Western buffer (25 mm Tris, 192 mm glycine, 20% methanol). RPA subunits were detected after incubation with anti-Rfa1 (24Brush G.S. Kelly T.J. Nucleic Acids Res. 2000; 28: 3725-3732Crossref PubMed Scopus (80) Google Scholar), anti-Rfa2 (24Brush G.S. Kelly T.J. Nucleic Acids Res. 2000; 28: 3725-3732Crossref PubMed Scopus (80) Google Scholar), or affinity-purified anti-Rfa3 polyclonal antibodies followed by horseradish peroxidase-linked goat anti-rabbit antibody (Pierce). Antiserum directed against Rfa3 as well as the affinity-purified preparation and pre-immune serum were generously provided by Steven Brill (Rutgers University). Ime2-His/Ha was detected after incubation with monoclonal antibody HA.11 (Covance) followed by horseradish peroxidase-linked goat anti-mouse antibody (Pierce). Protein bands were visualized by autoradiography using chemiluminescence reagents (Pierce). DNA Content Analysis—Cells were analyzed for DNA content by flow cytometry after staining with SYBR Green I (Molecular Probes) as described previously (25Brush G.S. Clifford D.M. Marinco S.M. Bartrand A.J. Nucleic Acids Res. 2001; 29: 4808-4817Crossref PubMed Scopus (23) Google Scholar). DNA content histograms were generated using WinMDI software. Immunoprecipitation—Stationary phase cells were harvested by centrifugation, resuspended in 0.1 m EDTA, 10 mm dithiothreitol (DTT) (2.5 ml/g cells), and incubated at 30 °C for 10 min. The cells were harvested by centrifugation again, resuspended in YPS (1% yeast extract, 2% bactopeptone, 1 m sorbitol) containing 200 μg/ml Zymolyase 100T (1 ml/g cells; U. S. Biochemical Corp.), and incubated at 30 °C for 2.5 h. The resulting spheroplasts were collected, washed twice with YPS, and then resuspended in lysis buffer (50 mm Hepes, pH 7.4, 100 mm KCl, 0.2% Tween 20, 1 mm DTT, 0.1 mm EDTA, 25 mm NaF, 100 μm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 1 μg/ml leupeptin). Resulting suspensions were incubated on ice for 30 min and then centrifuged at 12,000 × g for 15 min at 4 °C. Supernatants were collected and stored at -80 °C. For immunoprecipitation, lysate was incubated on ice for 2 h with antibody (HA.11, anti-Rfa1, or anti-Rfa3) or the respective control (no antibody, Rfa1 pre-immune serum, or Rfa3 pre-immune serum). Immune complexes were collected on EZview Red Protein G Affinity Gel (Sigma) by incubation at 4 °C for 2 h with gentle shaking and were subsequently washed once with lysis buffer containing 0.5 m NaCl and twice with lysis buffer alone. RPA Purification—RPA was purified from exponentially growing W303-1A (wild type) and YGB23 (rfa2-S122D) cells and from synchronously sporulating DSY1089 cells (wild type; 6-h time point). Purification over three columns (Affi-Gel Blue, ssDNA cellulose, and Mono Q) was performed based on methods described previously (19Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (205) Google Scholar, 42Sung P. Genes Dev. 1997; 11: 1111-1121Crossref PubMed Scopus (467) Google Scholar). Protein concentration was determined by the method of Bradford (43Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222179) Google Scholar) using bovine serum albumin as a standard. Relative levels of Rfa2 in the W303-1A and YGB23 RPA preparations were determined from a silver-stained gel (Fig. 4C) using a Kodak Digital Science Image Station 440CF equipped with 1D Image Analysis Software, version 3.5.3. Protein Kinase Assay—HA.11 immunoprecipitates were washed two times with kinase buffer (10 mm Hepes, pH 7.4, 10 mm MgCl2, 1 mm DTT) and then assayed for protein kinase activity in kinase buffer containing 10 μm ATP and 2.5 μCi of [γ-32P]ATP (25 μl final volume). Purified RPA (5.3 pmol) and M13mp18 single- or double-stranded DNA (17 fmol; New England Biolabs) were added where indicated. Reaction mixtures were incubated at 30 °C for 40 min and then subjected to 15% SDS-PAGE. Gels were dried either directly or after silver staining (Fig. 4C), and incorporation of radioactive inorganic phosphate into protein was detected by autoradiography and Storm PhosphorImager (Amersham Biosciences) analysis. Meiotic RPA Phosphorylation Requires Ime2—Our previous studies (25Brush G.S. Clifford D.M. Marinco S.M. Bartrand A.J. Nucleic Acids Res. 2001; 29: 4808-4817Crossref PubMed Scopus (23) Google Scholar) revealed that the middle subunit of yeast RPA (Rfa2) becomes phosphorylated in two separate reactions during meiotic progression. Because the primary reaction is induced early in meiosis when Ime2 is also known to be induced, we investigated Rfa2 status in wild type and ime2 mutant cells subjected to starvation conditions that induce entry into the meiotic program. As observed previously, two Rfa2 phosphoisomers were detected by Western blot analysis in wild type cells (Fig. 1A). A primary phosphoisomer was first observed early in meiosis prior to the bulk of pre-meiotic DNA replication (see Fig. 1B) and persisted throughout most of meiotic progression. A secondary hyperphosphorylated phosphoisomer, comprising only a small fraction of the total Rfa2, was induced later in sporulation. We demonstrated previously that generation of this phosphoisomer requires both Mec1 and initiation of recombination (25Brush G.S. Clifford D.M. Marinco S.M. Bartrand A.J. Nucleic Acids Res. 2001; 29: 4808-4817Crossref PubMed Scopus (23) Google Scholar). Deletion of IME2 abolished nearly all meiotic Rfa2 phosphorylation as determined by Rfa2 electrophoretic mobility (Fig. 1A). Consistent with previous reports (1Smith H.E. Mitchell A.P. Mol. Cell. Biol. 1989; 9: 2142-2152Crossref PubMed Scopus (150) Google Scholar, 3Yoshida M. Kawaguchi H. Sakata Y. Kominami K. Hirano M. Shima H. Akada R. Yamashita I. Mol. Gen. Genet. 1990; 221: 176-186Crossref PubMed Scopus (96) Google Scholar, 5Foiani M. Nadjar-Boger E. Capone R. Sagee S. Hashimshoni T. Kassir Y. Mol. Gen. Genet. 1996; 253: 278-288Crossref PubMed Scopus (63) Google Scholar), the ime2 mutant cells did not progress normally through pre-meiotic DNA replication, exhibiting a significant delay in the onset of DNA synthesis (Fig. 1B). In addition, these cells were incapable of forming mature asci at later time points (data not shown). Genetic studies suggest that Ime2 regulates pre-meiotic DNA replication by promoting phosphorylation and degradation of the B-type cyclin inhibitor Sic1 (14Dirick L. Goetsch L. Ammerer G. Byers B. Science. 1998; 281: 1854-1857Crossref PubMed Scopus (114) Google Scholar). To determine whether Ime2 indirectly activates meiotic Rfa2 phosphorylation by relieving Sic1-mediated inhibition of a cyclin-dependent kinase, we examined sic1 mutant and ime2 sic1 double mutant cells induced to enter meiosis. Deletion of SIC1 led to an accumulation of G2 cells that were incapable of entering the sporulation program (Fig. 2B) (14Dirick L. Goetsch L. Ammerer G. Byers B. Science. 1998; 281: 1854-1857Crossref PubMed Scopus (114) Google Scholar). As a result, a residual mitotic Rfa2 phosphoisomer was detected in sic1 and ime2 sic1 cells throughout the time course (Fig. 2A). In sic1 cells, two Rfa2 phosphoisomers were observed, whereas in ime2 sic1 cells, a single Rfa2 phosphoisomer was observed that did not appear to change in abundance relative to unphosphorylated Rfa2 during the time course. Therefore, deletion of SIC1 did not rescue the defect in Rfa2 phosphorylation conferred by deletion of IME2, arguing against the involvement of a B-type cyclin/cyclin-dependent kinase complex in catalyzing meiotic Rfa2 phosphorylation. It should be further noted that our ime2 sic1 double mutant did not appear to progress though meiosis as judged by flow cytometry (Fig. 2B) or formation of mature asci (data not shown). These results do not support the contention that a sic1 deletion can rescue the defect in ime2 cells that leads to defective meiotic progression. Ime2 Expression in Vegetative Cells Promotes RPA Phosphorylation—We further investigated the potential role of Ime2 as an RPA kinase by introducing IME2 into cells on a high copy 2-μm vector to allow for inappropriate mitotic expression of the meiosis-specific protein kinase. Cells expressing a His/Ha-tagged version of Ime2 (Ime2-His/Ha) exhibited two Rfa2 phosphoisomers during exponential growth, whereas cells expressing the kinase-dead version of tagged Ime2 (Ime2kd-His/Ha) exhibited the single Rfa2 phosphoisomer typical of mitotic cells (Fig. 3A, bottom panel, lanes 1 and 2). Expression of both forms of Ime2 was verified by Western blot analysis with antibody directed against Ha (Fig. 3A, top panel, lanes 1 and 2). The wild type Ime2 appeared to have slightly slower mobility than the kinase-dead version, possibly due to Ime2 autophosphorylation (see below). Our previous studies (23Brush G.S. Morrow D.M. Hieter P. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15075-15080Crossref PubMed Scopus (166) Google Scholar) revealed that Rfa2 phosphorylation observed during normal proliferation requires Mec1, and recent mapping studies have localized the site of Mec1-dependent phosphorylation to se
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