Copyright 0 1997 by the Genetics Society of America
Roles of Replication Protein-A Subunits 2 and 3 in DNA Replication Fork Movement in Saccharomyces cerm'siae
Hina S. Maniar, Richa Wilson and Steven J. Brill
Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855 Manuscript received September 12, 1996 Accepted for publication December 17, 1996
ABSTRACT Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (WAS and 3) that may cooperate with RPAl to bind ssDNA in a higher-order mode. To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an assortment of heat-lethal mutations in the genes encoding RPAQ and RF'AJ. At the permissive temperature, the mutants show a range of effects on DNA replication fidelity and sensitivities to UV and MMS. At the nonpermissive temperature, four outof five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block. In contrast, the fifth RPA2 mutant andall RPA3 mutants areable to complete at least one round of DNA replication at the nonpermissive temperature. The effect of these mutations on the stability of the RPA complex was tested using a coprecipitation assay. At the nonpermissive temperature, we find that RPAl and RPAQ are dissociated in the fast-stop mutants, but notin the slow-stop mutants. Thus, replication fork movement in vivorequires the association of at least two subunits of RPA. This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode.
EPLICATION Protein-A (RPA) is a three-subunit dues of the protein (GOMESand WOLD1995). In yeast, R single-stranded DNA bindingprotein that was RPAl was shown to contain two tandem copies of a 120- originally identified as a human protein required for amino acid domain with ssDNA binding activity. This SV40 DNA replication in vitro (WOBBEet al. 1987; FAIR- 120-amino acid region has weak sequence similarity to MAN and STILLMAN1988; WOLDand KELLY 1988). Hu- an Ecssb protomer and was termed an ssDNA binding man RPA (hsRPA) is composed of 70-, 34, and 11-kDa domain, or SBD (PHILIPOVAet at. 1996). subunits with a potent ssDNA binding activity and a The functions of the two smaller subunits of RPA are low affinity for dsDNA and RNA (KIM et al. 1992). In unknown. Neither subunitshows strong ssDNA binding Saccharomyces cmmisiae scRPA is composed of 69- (RPA1; activity, but both subunits are required for RPA func- encoded by RFAI), 36 (RPA2; encoded by RFA2), and tion invitro. For example, the p70 subunit of hsRPA 13-kDa(RPA3; encoded by REA?) subunits and each alone is unable to support SV40 DNA replication, and subunit is essential for viability (BRILLand STILLMAN antibodies to p34 inhibit thereaction (KENNY et al. 1990; 1989, 1991; HEYERet al. 1990). ERDILEet al. 1991).Further, hsRPA containing a C- . RPA is likely to be the eukaryotic homologue of the terminal truncation of p34 shows reduced activity in homotetrameric SSB of Eschm'chia coli (Ecssb). Like SV40 DNA replication (LEE andKIM 1995). Several ob- Ecssb, RPA is not only required for DNA replication in servations suggest a regulatory role for thep34 subunit. vitro, but is required for DNA repair (COVERLEYet al. The p34 subunit is subject to cell-cycle-dependent phos- 1991, 1992; GUZDERet al. 1995; HE et al. 1995; LEE phorylation (DINet al. 1990; DUTTAand STILIMAN1992; et al. 1995; LI et al. 1995; MATSUDA et al. 1995) and FOTEDARand ROBERTS1992) and immunolocalization recombination (HEYERand KOLODNER1989; MOORE et (CARDOSOet al. 1993; MURTIet al. 1996) as well as an al. 1991; FIRMENICHet al. 1995; SMITHand ROTHSTEIN interaction with the DNA repair protein XPA (LEEet 1995). Only the function of the large subunit (RF'A1) al. 1995; LI et al. 1995; MATSUDA et al. 1995) and in vitro has been clearly defined, however.RPAl has been phosphorylation by DNA-dependent protein kinase shown to bind ssDNA on its own (BRILLand STILI.MAN (BRUSHet al. 1994). Some of these studies indicate that p34 phosphorylation is not essential for SV40 DNA rep- 1989; WOLD et al. 1989; KENNY et al. 1990; ERDILEet al. 1991), andtruncation analysis revealed that the ssDNA lication or nucleotide excision repair (BRUSHet al. 1994; binding domain of hsRPAl lies withinthe first 441 resi- HENRICKSENand WOLD1994; LEEand KIM 1995; PAN et al. 1995), but is 'required to reverse the inhibitory effect of DNA-dependent protein kinase (HENRICKSEN Corre.$wndingauthor: Steven Brill, Department of Molecular Biology and Biochemistry, Rutgers University, 670 Hoes Ln., CABM, Piscata- et al. 1996). way, N1 08855. E-mail: [email protected] We have recently suggested that the minimal essential
Grnrtics 145 891-902 (April, 1997) 892 H. S. Maniar, R. Wilson and S. J. Brill regions of yeast RPA subunits 2 and 3 have sequence (BIULI.and STILLMAN1991) in pRS413 (SIKORSKIand HIETER similarity to the SBDs of RPAl and that, like the homo- 1989). Thefollowing 14 individual coding changeswere made via mutations in RFA2: H67A/H68A, D80A/H81A, E90A/ tetrameric Ecssb, the heterotrimer is composed of RPA D91A, R99K/K100A, E103A/D104A, D113A/D114A, K141A/ four SBDs (PHILIPOVAet al. 1996). RPA, like Ecssb, may E142A, K146A/K147A, H166A/H167A, K199A/D200A/ also have multiple ssDNA binding modes. In the case D201A, K219A/K220A, K225A/K226A, D244A/E245A, ofEcssb, two protomers bind 35 nucelotides (nt) of D266A/D267A. These 14 mutant fa2 plasmids were intro- ssDNA at low salt concentrations while four protomers duced into strain SBYIOS (Table 1) by selecting for histidine prototrophy. This yeast strain carries a chromosomal deletion bind 65-90 nt at higher ionic strength (LOHMANand of RFA2 and a wild-type copy ofREA2 in the centromeric OVERMAN 1985; LOHMANand FERRARI1994; RIGLER and URA3 plasmid, Ycp50 (Table 2) (PHII.IPOVAct nl. 1996). ROMANO1995). This binding mode causes compaction Transformants were streaked onto plates containing 5-fluoro- of the ssDNA due to higher-order binding or "wrap- orotic acid (FOA) (BOEKF,~t al. 1987) and tested for growth ping" about the tetramer (CHRYSOGELOSand GRIFFITH at 25" and 37". Only the E90A/D91A mutant Failed to grow at 37". Finally, random mutagenesis of REA2 and RZ-A? was 1982; BUJALOWSKIet al. 1988). In thecase of scRPA, we performed by mutagenic PCR essentially as described (LAXIN(; have shown that RPA2 can bind ssDNA as part of the et al. 1989). Plasmids pJM215 and pJM318 (BKIILand RPA trimer in a salt-dependent reaction similar to the STILLMAN1991) were individually subjected to 35 cyclesof higher-order wrapping of ssDNA about Ecssb (PHIL,- amplification using Taq DNA polymerase in the presence of 1 IPOVA et al. 1996). Wrapping of ssDNA has also been rnhf MnC12 and the universal forward and reverse sequencing primers. The PCR products were digested with BamHI and observed by electronmicroscropy (EM) ofscRPA SalI, ligated into the yeast centromeric plasmid pRS41.5, and bound to ssDNA (ALANI et al. 1992). While EM studies transformed into E. coli. The libraries (-100,000 clones each) of hsRPA bound tossDNA have also showna salt-depen- of mutagenized REA2 and RFA3 DNA were amplified in bacte- dent compaction of ssDNA, these investigators suggest ria and introduced into yeast strains SBY105 and SBYlll, that compaction results from structural changes other respectively, by selecting for leucine prototrophy. SBYl11 car- than wrapping of ssDNA about hsRPA (TREUNERet al. ries a chromosomal deletion of REA3 and a wild-type copy of the REA? gene in YCp50 (PHILIPOVAet al. 1996). Leucine 1996). While the biological significance of this mode prototrophs (-50,000 each) were replica-plated onto media of ssDNA binding is unclear, its conservation between containing FOA and placed at 37". Colonies that failed to such different organisms suggests that it is likely to be grow at 37" were recovered from the original transformation involved in some significant aspect of cellularDNA me- plate and the plasmid isolated in E. coli and rechecked for tabolism. temperature-sensitive growth in yeast. All rfn-t.? genes were then subcloned into the integrating To investigate the in vivo function ofRPA2 and RPA3, vector pRS403. RFA2 alleles were directed to integrate at the weisolated a numberof temperature-sensitive (Ts-) HZS3 locus of SBY105 by linearizing the pRS403 derivatives mutations in the genes for these subunits.Here we char- with NdeI, or MscI (fi2-ClOOand REA2wild-type). RFA3alleles acterize these allelesand show thatthey result ina range were directed to integrate at the HIS3 locus of SBYll1 by of phenotypes in DNA replication and repair. At the linearizing the pRS403 derivatives with NheI. Stable Ts- inte- grants were then obtained by selection on FOA. All strains nonpermissive temperature most,but not all, RPA2 mu- were grown at thepermissive temperature oF25" unless other- tants accumulate inthe G2 phase of the cell cycle while wise noted. RPA3 mutantsaccumulate primarily in G1. Further, Survival curves: Strains were grown in liquid WD medium most RPA2 mutants show a quick-stop DNA synthesis to early log phase and shifted to the nonpermissive tempera- phenotype while RPA3 mutants are able to complete ture (37") in a shakingwater bath. Aliquots of cells were taken at 0,2, 4,6, and8 hr after the temperatureshift and sonicated one round of DNA replication at the nonpermissive to disrupt cell clumps. Approximately 300 cells were then temperature. We demonstrate a strict correlation be- spread on 10-cm YPD plates and placed at 25". The number tween the quick-stop phenotype and dissolution of the of viable cells was determined by counting colonies after 3 RPAl/RPA2 complex. Thus, RPA2 is likely to play a days of growth. direct role in replication fork movement. Basedon our UV,MMS, and a-aminoadipate sensitivity: To measure sen- sitivity to ultraviolet (W) light, yeast strains were grown in current model of RPA, we conclude that higher-order YPD medium to early log phase and -500 cells spread onto ssDNA binding by RPA2 is essential for replication fork 10-cm YPD plates. The plates were then irradiated with the movement. indicated levels of ultraviolet light using a UV crosslinker (Stratagene). The number of surviving cells was determined by counting colonies after3 days growth at room temperature. MATERIALSAND METHODS Methyl-methanesulfonate (MMS)sensitivity was determined Isolation of RPA2 and RPA3 mutants: Conditional-lethal in liquid culture by adding MMS (Sigma) to a final concentra- mutations in the genes encoding RPA2 and RPA3 were iso- tion of 0.1% to exponentially growing cells in WD. At the lated using three approaches. First, a systematic truncation indicated times, aliquots were removed and neutralized with analysis of REA2 and REA3 indicated that the largest viable an equal volume of 10% sodium thiosulfate. The cells were truncations of these genes conferred growth at 30°, but not then washed with water and an appropriate volume (initially at 37" (PHILIPOVAet al. 1996). Second, clusters of charged containing 300 cells) spread onto YPD plates. The number amino acids within RPK2 were changed to alanine by site- of colonies was determined after 3 days growth at room tem- directed mutagenesis (KUNKEL1985) and screened for tem- perature. Mutator analysis was carried out by growing seven perature-sensitive growth as described (WEKTMANet al. 1992). independent cultures of each yeast strain to early log phase The template for this mutagenesis, pJM229, contains the wild- and plating 10" (or IO' for wild type) cells onto synthetic type RFA2cDNA and promoter on a PstI/XmnI fragment medium containing yeast nitrogen base without amino acids RPA2 and WAS in DNA Replication 893
TABLE 1 S. cerevisiae strains used in this study
Strain Genotype'' Reference
W303-la MATa ade2-l ura3-1 his3-11,15 trpl-1 leu2-?,112 cud-100 THOMAS and ROTHSTEIN (1989) SBYlO5 W303-la fa2-1::TRPl [pJM218 (REA2 URAJ)] PHILIPOVAet al. (1996) SBYll1 W303-la fa3-1::TRPl [pJM320 (RIA? URA?)] PHILIPOV.~rt al. (1996) HMY357 W303-la fa2-1::TRPl HlS3::RlCAz" This study HMY344 W303-la fa2-1::TRPl HIS3::fa2-55" This study I-IMY343 W303-la fa2-1::TRPl HIS3::fa2-21@ This study HMY345 W303-la fa2-1::TRPl HIL5'3::fd-214" This study HMY350 W303-la fa2-l::TRpI HIS3::fa2-215" This study HMY346 W303-la fa2-l::TRPl HIS3::fa2-C10@ This study HMY353 W303-la fa?-l::TRPl HlS3::fa3-313" This study HMY347 W303-la fa3-l::TRPl HlLY3::fa?-N7d' This study RW231 M'303-la fi2-l::TRPI rfn3-l::TRPl [pJM132 (REA1 RIA2 RFA3 URA3)] This study RS192 W303-la t@l-8 top2-l BRILL.and STERNGIANZ (1987)
~ ' Extrachromosomal plasmids are indicated by brackets. This strain has the indicated fa allele integrated at HIS?. or ammonium sulfate supplemented withlysine and other containing 0.1 mg/ml RNase A and incubated at 37" for 2- required amino acids, and 2 g/liter a-aminoadipate (Sigma) 4 hr. Propidium iodidewas then added to a final concentra- as the sole nitrogen source (CHATTOOet al. 1979). Plates were tion of 10 ,ug/ml and incubated at 4" for up to 1 week. At incubated at room temperature for 6 days and the number least 20,000 cells per sample were analyzed on a Coulter-Epics of colonies counted. The mutationfrequency was determined fluorescence-activated cell sorter. using the Method of the Median (LEAand COULSON1948). Immunoprecipitation and Western blot analysis: Yeast cul- The standard deviation of m was always <0.4m. tures were grown to early log phase and shifted to 37" at time Microfluorometricanalysis: Strains were grown in liquid zero. Ten-milliliter aliquots were taken at the indicated times WD medium to early log phase and shifted to the nonpermis- and crude protein extracts prepared as follows. Cells were sive temperature (37") in a shaking water bath. Aliquots of pelleted and resuspended in0.2 ml RIPA buffer (20 mM Tris cells were taken at 0,2,4, and8 hr after shift, washed with 1 ml pH 8.0, 0.15 M NaCl, 0.5% deoxycholate, 1% Triton X-100 50 mh1 sodium citrate, resuspended in 0.5 ml sodium citrate and 5 mM MgCI.,) and 0.1 ml dry glass beads were added.
TABLE 2 Plasmids used in this study
Name Reference Insert Vector
pJM2 15 WAY PSK BRILI.and STILLMAN(1991) pJM318 REA3 PSK BRILLand STILLMAN( 1991) pJM229 RFA2 pRS41 3b This study pJM245 REA2 pRS4 1 5 This study pJM331 REA3 pRS413 This study pJM337 REA3 pRS415 This study pJM 132 RIA l/REA2/RIA3 pRS416 This study pRT2 10 fa2-210 pRS415 This study pUT201-55 rfn2-55 pRS4 1 5 This study pRT2 14 fa2-214 pRS415 This study pRT2 15 fa2-215 pRS415 This study pDP2C100 rfi2-ClOO pRS415 PHII.IPOVAet al. (1996) pRT3 13 @3-313 pRS415 This study pDP3N70 fa3-N70 pRS415 PHII.IPO\"\ et al. (1996) Mp2-WT REA2 pRS403 This study Mp2-210 fa 2-2 10 pRS403 This study YIp2-55 fa2-55 pRS403 This study YIp2-2 14 fa2-21 4 pRS403 This study YIp2-2 15 fa2-215 pRS403 This study np2-~100 .fa2-C100 pus403 This study np3-3 13 fa3-313 pRS403 This study YIp3-N70 rJh3-N70 pRS403 This study pHM313 fa3-313 pRS413 This study pHM3N70 rfa3-N70 pRS413 This study "All RFM2 inserts are cDNA. pRS vectors have been described (SIKORSKIand HIETEK 1989). 894 H. S. Maniar, R. Wilson and S.J.Brill
Ycast cells were broken by vortexing (five times for 1 min, AXA alternating with 1 min periods on ice) and the supernatant 174 1 40 273 aa containing RPA was either immunoprecipitatedby incubation with anti-RPA2 antibody (BRII.1. and SrI1,I.~lIAN 1991) (1:100 RPA2 dilution) on ice for 1 hr followed by rocking with 50 pI of protein-A sepharose beads (I:] slurry in RIPA buffer) at 4" 1 70 122aa for 1 hr, or suhjected to simple Western blot analysis as de- RPA3 scribed (DIN r/ 90%) and thc appearance of schmoos. Cells were then pelleted, washed FI(;L'RE 1.-Mutant 1$12 and $n? allcles. (A) Schematic il- with Y medium and rcsnspcndcd in fresh Y mediumcon- lustration of the RPA2 and RPXS proteins. Regions of the taining 20 pCi/ml [.5,6]-"H rlracil (ICN). One portion of the proteins that are dispensable for viability are represented by cultrlrc was grown at 25", another at 37", and part of the 25" 0. (R) List of $02 and $I? alleles uscd in this study. Also culture was shifted to 37" after 90 min. The quality of the shown is the method by which they were created and the synchrony was verified microscopically: 78% of the arrested amino acid changes from wild-type. cells had small buds 90 min after relcase from the a-factor block. Aliquots (0.2 ml) were removed at the indicated times and placed in 0.5 ml stop solution (15% trichloroacetic acid, phenotype. Finally, the REA2 and WA3genes were sub- 200 pg/mI thymine, and 50 mbf sodium pyrophosphate) con- jected to PCR amplification under mutagenic condi- taining 0.1ml of unlabeledstationaly phase ycast cells as tions. Moderate levels of mutagenesis failed to result in carrier. Pelletcd cells were thcn resuspended in 50 p1 of 0.6 Ts- fa mutants, perhapsbecause of the large nonessen- \I NaOH and placed at 37" overnight for RNA hydrolysis. tial regions of these proteins, we increased the muta- Samples were neutralized with 6 \I HCl and precipitated with so 1 ml stop solution on ice for 1 hr. Precipitates were collected genesis by extending the number of PCR cycles. As a on glass fiber filters and the filters were washed, dried, and result, we identified several Ts- Vu2 mutants, three of the radioactivity determine by liquid scintillation counting. which were chosen for furtherstudy (Figure 1). We also identified a number of Ts- Vn3 mutants that displayed RESULTS similar phenotypes and one (Va3-313) was chosen for further characterization. As shown in Figure lB, these Isolation of conditional-lethal RFA2 and WA3 muta- PCR mutants contain multiple amino acid changes. tions: M'e had previously performedstructure/func- Each mutant gene was subcloned into pRS403 and tionstudies on RPA2 and RPAS in yeast and found integrated at the HIS3 locus to create a stable single- that both subunits contained large regions that were copy transformant (Table 1). Compared to strains har- dispensible for viability and essential regions that re- boringmutant Vi alleles on plasmids, thesestrains sembled the ssDNA binding domains (SBD) of RPAl showed noticeably lower levels of spontaneous rever- (Figure 1A).To determine thein vivo function of these sion. These strains were judged to be sufficient for ge- subunits a "plasmid shuffle" strategy and three meth- netic analysis, as they did not give rise to colonies when ods of in dromutagenesis of cloned DNA was used to streaked onto YPD medium and placed at the nonper- isolate seven conditional-lethal mutations in WA.2 and missive temperature (37"; Figure 2). At the permissive WA3 (Figure 1B). First, truncated alleles of WA2and temperature (25") all mutant strains grew noticeably REA3 that failed to grow at 57" were identified during slower than wild type (Table 3). the courseof the structure/function studies mentioned $22 and rju3 mutants show defects in DNA repair above (PIHILIPOVAr/ 01. 1996). The ~/ia2-C100allele cre- and DNA replication fidelity: To identi@ phenotypic ates a stop codon at residue 174 resulting in a trunca- differences behveen the rJi-l mutants, they were placed tion of 100 Gterminal amino acids from RPA2. The at the nonpermissive temperature for varying lengths @t3-N70 allele truncates 70 amino acids from the N- of time, and then plated at the permissive temperature terminus of RPAS by creating an initiating methionine to determine thefraction of viable cells. These survival at codon70. In the second methodwe used site-directed curves indicated a range of sensiti\ities, with many mutagenesis of IWA2 to change clusters of charged strains losing viability quicklv at 37" (Figure SA). The amino acidsto alanine, a techniquethat has been most severe killing was found in the $12-214and @2- shown to result in Ts- phenotypes in yeast (M~RTMAN C100 strains, which showed only 1% viability after 6 rt nl. 1992). Of these14 mutations, only the double hr at the nonpermissive temperature. Among the !-a2 point mutation ESOA, D9lA (@2-55) resulted in a Ts- mutant?, the ~$2-55and $72-210strains were the most RPA2 and RPX3 in DNA Replication 89.5 25OC 37OC WT WT
resistant, showing -30% viability after 6 hr. Of the two 1996). It is likely that the r/i/3alleles, rjk33'13and r/i/?- r/i/? mutants, r/ix3-l\VO was ven sensitive, losing over N70, are also defectivein NER given their slight sens.itiv- 90% viability in 6 hr, whereas $3-313 showed no de- ity to Lq'. crease in viability or increase in cell numberat the The ~fnmutants exhibit a wide range of sensitivity to nonpcrmissive tcmpcraturc (Figure SA). the alkylating agent MMS. As shown in Figure X,the Given the known reqtlirernentfor human RPA in +2-215 mutant is the most sensitive strain, ap DNA repair (COWRI ,EY rt d. 1991; HE PI d. 199.5; Lr rt proaching the sensitivity of the md2 mutant. A second d. 1995), we expected that the yeast rfi mutants might class of mutants (rfn3-313, fc12-210, and fu2-55) show show DNA repair defects at thepermissive temperature anintermediate sensitivity, while the fi2-CI00, $73- and tested them forsensitivity to DNA damaging agents. N70 and rfi2-214 mutants are essentially resistant to Most fW" .o 2 X 208 +214 I 0.4 IOP 1.4 a mutator phenotype. Interestingly, the two most ther- HW343 233 rf/12-210 2.2 2 0.7 X IO" 3.0 mosensitive mutations, Tjii2-CI00 and fi2-214, showed HW3.33 191 qiI3-313 3.2 -e 1.1 x lo-.' 4.4 HMY3.50 208 rfi12-215 3.4 2 1 .I x IO" 4.i a 10-fold difference in mutation frequency, with rf02- HMY346 183 @2-C100 8.0 ? 2.4 X IO" 11 214 showing high-fidelity replication that was nearly as HW3.50 224 $12-55 9.6 2 2.9 X 10. 13 good as wild type. The weakly Ts- fa3-313 displayed a rate fourfold greater than wild type. From the above I' Dorhling of optical tlcnsit!, in minutcs, in YPD at 2.7". assays we find that @i2-55is the most consistently defec- " 1,)Wmutation rate (prr cell per- gcncration) andstandard tlrviation. h;~setlon thc frcqltency ol'n-aminoadipatr-resistant tive allele, showing a relatively high rate of mutation rolonics. and high LV and MMS sensitivity. 896 H. S. Maniar, R. Wilson and S. J. Brill
Cell-cycle arrest of $a2 and $a3 mutants at the non- permissive temperature: To further characterize phe- notypic differences between the rfa mutants, we deter- "oo; A "oo; SURVIVAL at 37 'C h mined if cell growth arrested at a specific phase of the Y 8 100 cell-cycle at the nonpermissive temperature. Exponen- U 3 rfa3-313 tially growing strains were shifted to the nonpermissive L W temperature and aliquots were taken at 2-hr intervals rfa2-55 i? and fixed for microfluorometric analysis. As shown in -.3 rfa2-210 2 10 Figure 4, several 7322 mutants show preferential cell- rfa2-215 cycle blocks. Most $a2 mutants (-210, -55, -215) accu- 5 rfa3-N70 mulate primarily in the G2/M phase of the cell cycle as judged by this analysis. However, two mutants, $12- 1 - 214 and rfa2-ClO0, accumulate predominantly in G1. 0 2 4 6 8 Time (Hours) In the case of rfa2-CIOO there is a substantial G2/M population at6 and 8 hr after theshift to nonpermissive temperature. Thiseffect is unlikely to be due to IntDNA as very similar profiles were obtained when po deriva- tives of these strains were analyzed (data not shown). To confirm the above cell-cycle arrest points, we ob- served the cell morphology of the mutants after8 hr at the nonpermissive temperature. As shown in Figure 5, strains judged to arrest primarily in G2 by microfluor- ometric analysis (fk2-210, -55, -215) arrest with a sig- nificant proportion of budded cells, indicative of a late S-phase or G2/M block. The ?fa2 mutants that were judged to accumulate in G1 (-214, -CIO0) both arrest primarily as unbudded cells. We conclude that there are 0 40 80 120 160 200 at least two classes of ?fa%mutants that display different Dose (J/& teminal phenotypes. C Both .f;z? mutants arrest at the nonpermissive tem- perature primarily in the G1 phase of the cell cycle, but with significant G2/M populations, as judged by microfluorometric analysis (Figure 4). Like the fu2- ClOOstrain, both fa? mutants display an initial increase in the G2/M population, followed by an increase in G1 populationat late times afterthe temperature shift. Consistent with these results, rfn?-N70 cells arrest pre- dominantly as unbudded cells while most qa?-~-313cells arrest with very small, or no buds (Figure 5). Effect of the rfa mutations on the stability of the RPA complex: We suspected that the rapid Ts- pheno- 0 2 4 6 8 10 type of the rjiL mutants could be due to one of three Time (Hours) events. In the first case, mutant RPAP or WAS subunits might partially denature at thenonpermissive tempera- FIGVRE3.-Phenotypic analysis of fa2 and fa3 mutants. ture, leaving the trimeric complex intact but disrupting (A) Survival at 37". Strains were grown to early log phase in essential interactions between the subunits, or between WD broth at E", incubated at 37" for the indicated times, RPA and otherfactors. In the secondcase, mutant RPA2 and the percentage of viable cells determined by plating cells or RPAS subunits might denature and become sepa- on WD medium and counting the number of colonies after rated from the RF'A trimer at the nonpermissive tem- 3 days growth at 25". (B) UV sensitivity. Strains were grown to early log phase in WD broth, spread ontoWD plates, and perature, leaving the other subunits nonfunctional. Fi- immediately irradiated with the indicated dosage of UV light. nally, mutant RPAP or WAS subunit might denature Plates were incubated in the dark for 3 days at 25" and the at the nonpermissive temperature, leading to degrada- percentage of viable cells determined by counting the num- ber of colonies. (C) MMS sensitivity. Strains were grown to early log phase in WD broth and incubated with 0.1% MMS HMY353 (rjU3-?13), and HMY357 (WA2REA?). Each experi- for theindicated times before neutralization. The percentage ment was performed at least twice and each data point was of viable cells was determined as in A. Strains are HMY343 measured in duplicate. A minimum of two dilutions were used (fa2-210),HMY344 (fk2-55),HMY345 (fu2-214),HMY346 in A and C. Thc figures present the averages obtained from (rJilZ-Cl00) , HMY347 (rfrc?-N70), HMY3.50 (~fa2-215), all experiments. RPXL and RPA3 in DNA Rcplication 8%
FI(A.I~I:..5.-Tern~it1aI phenotypr ol. r/u? ;rnd 1ju3 strains. Each 01' thc strains H.1IY3-13 (+/2-210), H!vlY344 (#2-57), HW34.5 ($/2-214), IH\4Y:%46 (r///2Xlf)f)),HMY34i (+3- .VX)),HMY:WI ($/2-215), HMW?~(qi(3-313). ant1 HXnrsi (fWA2 IUd3) was grown to carly log phasr in YPI) hroth at 2.5," and shilied to :47". Samples werc t;~kenat 8 hr after shift, fixed and cxamined hy phase contrast microscopy. Magnifica- tion, 6OOX.
Extracts were made from cultures grown at 2.5" and 97" and RPA was immunoprecipitated with RPA2 antise- rum followed by Western hlottingwith RPAl antiserum. Wild-type cells showa strongp69 IP-Western signal that increases after 2, 4, and 8 hr at 37" (Figure 6, top). This rise in signal is probably due to the increase in cell nwnber during the course of the cxperiment. As ex- pected, the p69 subrunit in these wild-type extracts is FI(AXI<4.-~icroflr10romrtric analysis of rji/2and $13 rnu- stable at 37" as.jltdgetl by a simple \Vestern blot (Figure tmt strains. Each of the strains H1W343 (rfi/2-210),HMY344 (5, bottom). In contr~st,the following four mutantsshow ($/2-77), HMY34.5 (r/,t2-214).HMY34i (~ji/2-C100),HW347 reduced $9 IP-M'esternsignal atthe nonpermissive (t$/3-.V70), HW3.50 (cji/2-215), HW3.53 (rji/?-313),and temperature: @2-55, -210, -214, and -CIOO. In three of HMY35i (1W\2 1<1<.\3)was grown to wrly log phasc in YPD hroth at 2.5" and shifted to 3'7". Samples WCIT taken at the these m~~tants(~ji(2-55, -210, -214), the p69 srlbtlnit was intlicatrdtimes, fixed, stained with propitlium iodide and jrdged to be stable by the parallel p69 Western blot counted hy FACS. (Figure 6). Further, probing the IP-Western blot with RPA2 antiserum indicated that the mutant $36 protein tion of thc other subunits, including the known ssDNA in these three strains IVils present and immunoprecipi- binding subrmit, RPAl. To distinguish among these tated at the nonpermissive temperature, verifi.hg that possibilities we examined the effect of the shift to non- it I1ad not degraded (data not shown). Therefore, in permissivc temperature on the stability ofthe RPA com- these three mutants the RPAl/RPA2 complex is weak- plex using immunoprecipitation and Western blot (IP- ened or dissociated at the nonpermissive temperature M'cstern) analvsis. (case 2, >1bo\.t.: simple complex dissociation). In the H!)X 14. S. Maniar. R. "ilson ;~ndS..J. Brill
DNA synthesis profiles indicatethat each of the mutant cultures display normal kinetics of DNA syn- thesis at the permissive temperature (Figure 7). Ini- tii~lly,thcrc is it lag phase during which the cells re- cover from a-factor arrest and enterG1. The synthetic phase then appears as a sharp incrcase in incorpo- rated t-adioactivity followed by a plateau andthen ;~nothcrround of DNA synthesis. Daring the first S- phase, 10,000- 16.000 cpm of:'H-prccursor ilrc incor- porated into DNA in -3 hr. In cotltrilst, whc~nsome of the mutant strainsare released directly at 3f", only 21 small i1moutlt of DNA synthesis is ohsrtTed (Figure c 1). Using this protocol the rfn2-210, -55, -214, and - CIOO mutantstrains incorporate only 1000-4000 cpmover the co~trscof theexperiment, some of which is due to mtDNA replication. The lack of DNA synthrsis could he due to defects in initation, clonga- tion, or both. To distinguish hetwccn thcsc possibli- ties, a shift to 3f" \\'as performed within S-phase. Upon shift to Sf", these four tjh2 mutants abruptly Ft(;t'ttt< (i.-Stat)ilit\. 01' tl~rl
32000 B rfa2-55
0 ""-1 D rfa2-215 0
, G rfa3-3iV70
0 1 2 3 4 5 6 0 1 2 3 4 5 6 Time (Hours) Time (Hours)
FIGURE 7.-DNA synthesis profiles. Each of the indicated strains was grown to mid log phase in Y minimal medium at 25", arrested with a-factor and released into medium containing[5,6]-'HH-uracil under thefollowing conditions. One sample was released and maintained at 37" (0-0)and a second sample was released and maintained at 25" (0-0).After 1.5 hr (arrow) a portion of the 25" sample was shifted to 37" (m - - - m). Aliquots were taken at the indicated times and the number of alkali-resistant TCA- precipitable counts determined. Strains are asfollows: A, HMY343 (rfa2-210);B, HMY344 (fu2-55);C, HMY345 (rfa2-214);D, HMY350 (fu2-215);E, HMY346 (fuZ-CI00);F, HMY353 (rfa3-313);G, HMY347 (fu3-N70);H, RS192 (topl-8 to@!-I). complemented by a single URA3 plasmid containing the transformants onto medium containing FOA. Al- each of the wild-type REA genes. We transformed most all conditional alleles of REA2 were inviable in RWY231 with all pairwise combinations of fu2 IXU2 strains harboring the fa3-313 or fa3-N70 mutations, plasmids and rfa3 HIS3 plasmids (Table 2) and streaked suggesting that RFA2 and REA3 may be partially redun- 900 H. S. Maniar, R. Wilson and S. J. Brill dant (data not shown). Only in a single case, the fu2- phase that are refractory to repair upon return to the 214 fa?-?I? transformant, was the double mutant via- permissive temperature. ble. It is likely that these two alleles are functionally Our data implicate both RPA2 and RPA3 in the pro- distinct and capable of producing mutant proteins that cessof DNA repair. Most Vu2 and fu? mutants dis- are together able to make essential interactions at the played an increased sensitivity to DNA damaging permissive temperature. agents, but allele-specific differences in the degree of sensitivity and a selective response to the two agents DISCUSSION suggest that specific repair pathways may be affected in the various mutants. For example, thefu?-?l? and fu2- We report theisolation and characterization of condi- 215 alleles are only moderately UV-sensitive but display tional-lethal mutations in REA2 and REA?, which en- the greatest sensitivity to MMS, while two other moder- code the two small subunits of Replication Protein-A. atelyUV-sensitive alleles, fu2-Cl00 and fa?-N70, Based on the cell-cycle-dependent phosphorylation of showed no increased sensitivity to MMS. This repair RPA2 and the absence of any biological activity, these defect has been confirmed biochemically for some al- subunitsare thought to be regulatory proteinsthat leles. Nucleotide excision repair in vitro is reduced in might control the ssDNA binding activity of the largest extracts made from fu2-210 and fu2-215 strains and subunit, RPAl (DUTTA and STILLMAN1992; FOTEDAR this defect can be complemented by the addition of and ROBERTS1992; CARDOSOet ul. 1993). With the re- exogenous wild-type RPA (HE et al. 1996). The rfa2 cent identification of potential ssDNA binding domains and $3 alleles that show specific repair defects should in these subunits, it is possible that they mayplay a permit a genetic analysis to identify interacting compo- direct role in binding ssDNA during DNA replication nents in these repair pathways. (PHILIPOVAet nl. 1996). Our results describing the ef- A number of phenotypes that we examined in the fects of mutations affecting these subunits strengthen Vu2 and fa? mutants displayedallele-specific differ- this hypothesis. ences. First, fa2-215 was unique among the fa2 mu- Our data indicate that RPA2 is essential for the elon- tants in that it did not display a fast-stop replication gation phase of DNA replication. DNA synthesis studies phenotype and was able to complete two rounds of in synchronized cultures revealed that most conditional replication at thenonpermissive temperature. We think alleles of REA2 cause an immediate cessation of DNA it is unlikely that fa2215 is leaky given its rapid loss of synthesis atthe nonpermissive temperature (a “fast- viability at 37”. We suspect that the lethal defect in this stop” phenotype) reminiscent of the block to elonga- mutant is similar to that of the fu? mutants: poor fidel- tion seen in the absence of a topological swivel (BRILL ity of replication is exacerbated at37”, leading to exces- et ul. 1987; KIM and WAN(:1989). Although our experi- sive DNA damage that, in this case, arrests in G2. Sec- ments do not directly address the initiation phase, no ond, while DNA replication mutants are expected to DNA synthesis was observed when these strains were arrest at this phase of the cell cycle, not all mutants released from G1 arrest directly at the nonperrnissive showed this terminal phenotype. The fa2-214 and fu2- temperature. This result is consistent with a require- ClOO are fast-stop mutants, but arrest with signficant ment for RPA2 in initiation, but it might also be ex- G1 populations, like the fu? mutants. How these mu- pected in the absence of any significant elongation. tants are able to escape the G2 checkpoint is unclear, Further studies will be needed to confirm an in vivo but one possibility is that RPA2 and/or WAS is part of a role for RPA2 and RPA3 in the initiation of DNA repli- G2 signalling pathway that is defective in these mutants. cation. In contrast to these fu2 alleles, neither fu? Finally, the fu2-214 allele is unique in that it displays allele had a drastic effect on theinitiation or elongation a fast-stop phenotypeat the nonpermissive tempera- ofDNA synthesis at the nonpermissive temperature. ture, but essentially wild-type fidelity at the permissive The fu? mutants initiated and completed one round temperature. In this mutant, the roles of RPA2 in pro- of DNA replication at the nonpermissive temperature moting replication fork movement and replication fi- before synthesis terminated (a “slow-stop” phenotype). delity appear to be separated. At late times fa? mutant cells were found primarily in While this work was in progress, a report describing the G1 phase of the cell cycle. While it is possible that two fu2Ts- alleles appeared (SANTOCANALEet al. 1995). the fa? mutants are defective in the initiation of DNA By coincidence, the amino acid changes in these two replication in the subsequent cell cycle, we believe this alleles (D91G and C173-STOP)closely resemble the is unlikely given their rapid loss of viability at 37” and changes in our rfu2-55 and fu2-CIOO alleles, respec- their ability to initiate and complete the first round of tively, We find essentially identical phenotypes in our synthesis at the nonpermissive temperature. Why then mutants, except that the fu2-ClOO allele accumulates are these strains inviable?We suggest that these mutants primarily in G1 at the nonpermissive temperature, as accumulate incomplete replication products at thenon- opposed to the fu2-C17?-STOP allele, which blocks at permissive temperature and escape the G2 checkpoint G2/M. This inconsistency may be due to experimental to be recognized by a G1 checkpoint. Certain errors, differences such as the time of incubation at the non- such as double-stranded breaks, might occur during S permissive temperature, orto strain background differ- RPA2 and RPA3 in DNA Replication 90 1 ences. The additional alleles that we have identified tion properties of y-RPA, a yeast single-strand-DNA-binding pro- tein. J. Mol. Biol. 227: 54-71. allowed us to test how RPA2 and RPA3 affect the stabil- BOEKE,J. D., J. TRLEtlEART, G. NATSOL~I.ISand G. R. FINK,1987 5- ity of the RPA complex and the elongation phase of fluoroorotic acid as a selective agent in yeast molecular genetics. DNA replication. Methods Enzymol. 154: 164-175. BRII.I.,S. J., and B. SIII.I.MN, 1989 Yeast replication factor-A func- We were struck by the correlation between the insta- tions in the unwinding of the SV40 origin of DNA replication. bility of the RPAl/RPA2 complex in fast-stop mutants Nature 342: 92-95. and its stability in slow-stop mutants. We have shown BKIIL, S..J., and B. STII.I.MAN,1991 Replication factor-A from Sur- charomyrrs cermisiar is encoded by three essential genes coordi- that while the RPAl subunit is present in three fast-stop nately expressed at S phase. Genes Dev. 5: 1589-1600. mutants (rfa2-55, rfa2-210, -C214) at the nonpermissive BRIIL, S.J., S. DINARDO,K. VOF,I,WI.-MEI.MANand R. STLKNGIANZ, temperature, it is poorly precipitated along with RPA2 1987 Need for DNA topoisomerase activity as a swivel for DNA replication for transcriptionof ribosomal RNA. Nature 326: 414- subunit (Figure 6). We suggest that this result is due to 416. a weakened or unstable RPAl/RPA2 complex at the BRLISH,6. S., C. W. ANDERSONand T.J. &I.IS, 1994 The DNA-acti- nonpermissive temperature, since we know that p36 is vated protein kinase is required for the phosphorylation of repli- cation protein A during simian virus 40 DNA replication. Proc. present and recognized by the antiserum under these Natl. Acad. Sci. USA 91: 12520-12524. conditions. In the case of rfa2-CI00, the p69 subunit B[J~.UOWSKI,W., L,. B. OVERMANand T. M. L,OHIVWN,1988 Binding degrades at the nonpermissive temperature, clearly re- mode transitions of Escherichia coli single strand binding pro- tein-single-stranded DNA complexes. Cation, anion, pH, and sulting in loss of RPA activity. Interestingly, the fast-stop binding density effects. J. Biol. Chem. 263: 4629-4640. DNA synthesis phenotype of this mutant is no more CAROOSO,M. C.,H. LEONHARDTand B. NAr)Al.-GlNARl), 1993 Rever- severe than any other fast-stop mutant in which RPAl sal of terminal differentiation and control of DNA replication: cyclin A and Cdk2 specifically localize at subnuclear sites of DNA is not degraded (Figure 7). Finally, three TsC strains replication. Cell 74: 979-992. (rfu2-215, fa?-N70, and fa?-313) have a stable RPA1/ CHATTOO,B. B., F. SHkXh4AN, D.A. ALVB.U.IS, T. A. Fpx.1.s-rEo.1. D. RPA2 complex at the nonpermissive temperature and MEHNERTel al., 1979 Selection of (ys2 mutants of the yeast Surchuromycrs crrmisiar by the utilization of a-aminoadipate. Ge- no defect in the elongation phase of replication. netics 93: 51 -65. While it is possible that RPA becomes unstable in the CIIRYSOGEI.OS,S., and J. GRIFFITH,19F2 E,\chm'chiu coli single-strand presence of a replication fork block, we believe that the binding protein organizes single-stranded DNA in nucleosome- like units. Proc. Natl. Acad. Sci. USA 79: 5803-5807. simpler interpretation of our results is that some Ts- CO\'EM.EY, D., M. K. mNw,M. MYNN,W. D. RYpp, D. P. LANk. rl al., mutations cause RPAl and RPA2 to dissociate, resulting 1991 Requirement for thereplication protein SSB in human in a block to replication fork movement. Interestingly, DNA excision repair. Nature 349: 538-541. COVERIXY,D., M. K. KENNY, D.P. LANE and R.D. WOOD, 1992 A both of these subunits are proposed to bind ssDNA role for the human single-stranded DNA binding protein HSSB/ (PHILIPOVAet al. 1996). While the role of the multiple RPA in an early stage of nucleotide excision 1-epair.Nucleic Acids ssDNA binding subunitsin RPA is unknown, we suggest Res. 20: 3873-3880. DIN, S.,S. BMI.~.,M. P. FAIRhIAN and B. STII.I.~IAN,1990 Cell-cycle- that the interaction of ssDNA with both subunits is es- regulatvd phosphorylation of DNA replication Factor A from hu- sential for replication fork movement. One possibility is man and yeast cells. Genes Dev. 4: 968-977. that the multiple ssDNA binding domains wrap ssDNA DL~TTA,A,, and B. sTIl.l.h5AN, 1992 cdc2 family kinases phosphory- late a human cell DNA replication factor, RPA, and activate DNA about the RPA trimer in a higher-order mode during replication. EMBO J. 11: 2189-2199. replication elongation. Such an effect is observed with ERDII.E,L.F.,W. D. 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Biol. 15: 1620- 1631. cules to different sites on the DNA so as to increase its FOTEDAR,R., and J. M. ROHEKIS,1992 Cellcycle regulated phos- effective localconcentration. Either modelis consistent phorylation of RPA-32 occurs within the replication initiation with the requirement for multiple ssDNA binding do- complex. EMBO J. 11: 2177-2187. GOMF.~,X. V., and M. S. WOI.U, 199.5 Structural analysis of human mains in all known cellular SSBs (bacterial SSB, mtSSB, replication protein A. Mapping functional domains of the 70- and RPA). Further studies of the RPA mutants in yeast kDa subunit. J. Biol. Chem. 270: 4.534-4543. will be of immense value in distinguishing between GUZDEK, S. N., Y. H~RRAKEN,P. SYNC:, L. PRAKASIIand S. PMKASH, 1995 Reconstitution ofyeast nucleotide excision repair with these alternative models. purified Rad proteins, replication protein A, and transcription The authors thank MI^ HAMPSWand JAK MULLENfor comments factor TFI1H.J. Biol. 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