Dominant Mutations in Three Different Subunits of Replication Factor C Suppress Replication Defects in Yeast PCNA Mutants

Neelam S. Amin, K. Michelle Tuffo and Connie Holm Department of Pharmacology, Division of Cellular and Molecular Medicine, University of California, San Diego, California 92093-0651 Manuscript received April 23, 1999 Accepted for publication August 25, 1999

ABSTRACT To identify proteins that interact with the yeast proliferating cell nuclear antigen (PCNA), we used a genetic approach to isolate mutations that compensate for the defects in cold-sensitive (CsϪ) mutants of yeast PCNA (POL30). Because the cocrystal structure of human PCNA and a p21WAF1/CIP1 peptide shows that the interdomain region of PCNA is a site of p21 interaction, we specifically looked for new mutations that suppress mutations in the equivalent region of yeast PCNA. In independent screens using three different CsϪ mutants, we identified spontaneously arising dominant suppressor mutations in the RFC3 gene. In addition, dominant suppressor mutations were identified in the RFC1 and RFC2 genes using a single pol30 mutant. An intimate association between PCNA and RFC1p, RFC2p, and RFC3p is suggested by the allele-restricted suppression of 10 different pol30 alleles by the RFC suppressors. RFC1, RFC2, and RFC3 encode three of the five subunits of the replication factor C complex, which is required to load PCNA onto DNA in reconstituted DNA replication reactions. Genomic sequencing reveals a common region in RFC1p, RFC2p, and RFC3p that is important for the functional interaction with PCNA. Biochemi- cal analysis of the wild type and mutant PCNA and RFC3 proteins shows that mutant RFC3p enhances the production of long DNA products in pol ␦-dependent DNA synthesis, which is consistent with an increase in processivity.

HE proliferating cell nuclear antigen (PCNA) is an fore, the interaction of PCNA and proteins of the RFC Tessential factor in eukaryotic DNA replication and complex may play a crucial role in the execution of repair processes, and it may also be an important target DNA synthesis. PCNA has been shown to function in for coordinating cell-cycle regulation with DNA replica- many DNA repair processes, such as base and nucleotide tion (reviewed in Jonsson and Hubscher 1997 and excision repair (Nichols and Sancar 1992; Shivji et Kelman 1997). During DNA replication, PCNA mark- al. 1992; Matsumoto et al. 1994; Frosina et al. 1996), edly enhances the efficiency of incorporation of nucleo- RAD6-dependent error prone repair (Torres-Ramos et tides into DNA by securely attaching the DNA synthesis al. 1996), DNA methylation (Chuang et al. 1997), and machinery onto the DNA (reviewed in So and Downey DNA mismatch repair (Johnson et al. 1996; Umar et al. 1992; Wyman and Botchan 1995; Jonsson and Hub- 1996). That PCNA might play a role in DNA mismatch scher 1997; Kelman 1997). In vitro DNA replication repair is particularly intriguing because mutations in reactions show that PCNA is first loaded onto the DNA known human mismatch repair genes, such as hMSH2, by a complex of five proteins called replication factor hMLH1, and hPMS2, have been linked to colorectal C (RFC) in an ATP-dependent reaction (Lee et al. 1988; cancers (Liu et al. 1996). Lee and Hurwitz 1990; Tsurimoto and Stillman Apart from its role in DNA replication and DNA 1991a; Yoder and Burgers 1991; Fien and Stillman repair, mammalian PCNA is also the target for the bind- 1992; Podust et al. 1995). Next, PCNA binds to the ing of the cyclin-dependent kinase (CDK) inhibitor DNA polymerase (␦ or ε), and DNA elongation occurs p21WAF1/CIP1 (Xiong et al. 1992, 1993; Zhang et al. 1993; processively (Lee and Hurwitz 1990; Burgers 1991; Flores-Rozas et al. 1994; Waga et al. 1994), which is Lee et al. 1991b; Tsurimoto and Stillman 1991b; induced under conditions of DNA damage in a p53- Podust et al. 1992). Because RFC is also able to unload dependent manner (El-Deiry et al. 1993). Although a PCNA from the DNA, PCNA may be shuttled onto and p21 homolog has not yet been identified in yeast, the off of Okazaki DNA fragments during lagging-strand striking similarities in the crystal structures of human DNA synthesis (Yao et al. 1996; Cai et al. 1997). There- and yeast PCNA suggest that there may be similarities in their regulation as well. Biochemical studies show that p21 blocks DNA synthesis by binding to PCNA (Flores-Rozas et al. 1994; Waga et al. 1994) and that Corresponding author: Connie Holm, Department of Pharmacology, the C-terminal 22 amino acids of p21 are sufficient to Division of Cellular and Molecular Medicine, University of California, 9500 Gilman Dr., San Diego, CA 92093-0651. inhibit PCNA activity (Warbrick et al. 1995). The deter- E-mail: [email protected] mination of the cocrystal structure of human PCNA and

Genetics 153: 1617–1628 ( December 1999) 1618 N. S. Amin, K. M. Tuffo and C. Holm the C-terminal p21 peptide has identified the interdo- For flow cytometry experiments, rho0 strains were created by ϩ main and interconnector loop regions of the PCNA growing rho strains CH2165 (POL30), CH2161 (pol30-104), and CH2392 (pol30-104 RFC3-3) in YEPD (see below) con- monomer as the site of interaction of the p21 peptide taining 25 ␮g/ml ethidium bromide as outlined in Sherman (Gulbis et al. 1996). However, this structure leaves open et al. (1986). A list of all strains used in this study is presented the question of the mechanism of inhibition of DNA in Table 1. synthesis. While it is possible that p21 binding affects YEPD (rich) and SD (synthetic dextrose) media were used the monomer-trimer ratio of PCNA in the cell, it appears to grow yeast cells. YEPD medium contains 1% yeast extract, 2% bactopeptone, 2% dextrose, with the presence or absence more likely that p21 interferes with the binding of essen- of 2% bactoagar. SD medium contains 0.67% yeast nitrogen tial DNA replication proteins to PCNA. Candidates for base, 2% dextrose, and 2% bactoagar. SC (synthetic complete) these essential proteins include DNA polymerases ␦ or medium contains 60 mg of leucine, 30 mg of lysine, and 20 ε, or subunits of RFC. mg of uracil, adenine, histidine, and tryptophan in 1 liter of We have previously used the Saccharomyces cerevisiae SD medium. For MMS-containing plates, MMS (Sigma, St. Ϫ Louis) was added to autoclaved YEPD at a final concentration PCNA gene to identify cold-sensitive (Cs ) mutations of 0.01, 0.015, or 0.02% prior to pouring plates. Sporulation that affect the interdomain region of the yeast PCNA medium contains 1% potassium acetate, 0.1% yeast extract, protein structure (Amin and Holm 1996). In vivo analy- 2% bactoagar, and 0.05% dextrose. sis of the CsϪ pol30 mutants suggests that the interdo- Pseudoreversion screen: To obtain pseudorevertants of main region of the PCNA monomer is important for pol30 mutants, we selected spontaneously arising suppressors of three different cold-sensitive PCNA mutations. Specifically, both efficient DNA replication and for repair of methyl 10–33 independent cultures of strain CH2159 (pol30-100), methanesulfonate (MMS) and UV-induced DNA dam- CH2161 (pol30-104), or CH2171 (pol30-108) were grown over- age (Amin and Holm 1996). A comparison of the yeast night in YEPD at 30Њ. Approximately 107 cells derived from and human PCNA structures reveals that they are virtu- each of the pol30 mutant cultures were spread on each YEPD ally superimposable (Krishna et al. 1994; Gulbis et al. plate and incubated at the restrictive temperature of 14Њ or the semipermissive temperature of 20Њ. The use of two different 1996). Thus, it is striking that the p21 peptide makes temperatures was initially intended to enhance the specificity contacts with the interconnector loop and the interdo- of the screening process. Spontaneously arising revertant colo- main region of human PCNA, the same region in yeast nies were selected and retested for loss of their CsϪ and Mmss PCNA where our CsϪ mutations are located (Krishna phenotypes. In addition, the revertants were also examined et al. 1994; Gulbis et al. 1996). Furthermore, because for the appearance of new phenotypes, including heat sensitivity or sensitivity to hydroxyurea. cold sensitivity often affects protein-protein interactions Genetic analysis of intragenic and extragenic suppressors: (Cantor and Schimmel 1980; Strauss and Guthrie To determine if suppression of pol30 mutant phenotypes is 1991; McAlear et al. 1994), it is likely that the interdo- conferred by a single gene, the suppressed pol30 strains were main region of PCNA is a key region for protein-protein crossed to a CsϪ strain (CH2159, CH2161, or CH2171) bearing interactions. the same pol30 allele as present in the pseudorevertant. Exami- To identify proteins that interact with the interdomain nation of the phenotype of the diploid strain revealed whether suppression is dominant or recessive. Because the diploid region of yeast PCNA in vivo, we looked for suppression strains were homozygous for the pol30 mutant allele, 2:2 segre- Ϫ of the defects of three Cs pol30 mutants by spontane- gation of the Csϩ phenotype was indicative of a single gene ously arising mutations in other genes. We obtained conferring suppression upon tetrad analysis. Further analysis both intragenic and extragenic suppressors that are of 24 strains carrying single-gene suppressors was carried out dominant for suppression. In independent screens with to determine whether suppression was due to intragenic or extragenic mutations. The revertant strains were crossed with three different pol30 alleles we obtained extragenic sup- a POL30 strain (CH2237), and tetrad analysis revealed that pressors that affect the RFC3 protein. Additionally, we 12 of the 24 revertants were likely to contain intragenic muta- obtained mutations in RFC1 and RFC2 that suppress the tions; genomic sequencing was used to confirm that the muta- defects of one of the pol30 alleles. Our results suggest tions were in the POL30 gene (mutations and amino acid that RFC1p, RFC2p, and RFC3p are important for a changes described below) and not the neighboring RFC5 gene on chromosome II. functional interaction with the interdomain region of To determine the number of different genes represented yeast PCNA. In particular, we have identified an evolu- among the remaining 12 extragenic suppressors, we crossed tionarily conserved region in RFC1p, RFC2p, and each of the MATa suppressor strains with one or more of the RFC3p that is important for the functional interaction. MAT␣ suppressor strains and performed tetrad analysis. The Furthermore, biochemical analysis of the wild-type and suppressor genes were initially referred to as SOP1, SOP2, mutant PCNA and RFC3 proteins shows that the sup- SOP3, etc., for suppressor of pol30. To identify the gene product encoded by the SOP genes, we tested whether any of the sop pressor RFC3p enhances the production of long DNA genes were candidates suggested from biochemical studies. products in polymerase ␦-dependent DNA synthesis. Because all of the extragenic suppressors of the pol30-104 mutation affected a single gene (SOP1), we crossed a single extragenic suppressor strain CH2386 (pol30-104 SOP1-1) with MATERIALS AND METHODS strains containing markers to each of the genes encoding subunits of the RFC complex. These strains are CH2237 Strains and media: All yeast strains mentioned in this study (RFC1.URA3), CH1785 (ira2::HIS3, which is linked to RFC4), have an S288c strain background, and they were constructed CH756 (cdc8-1, which is linked to RFC2), CH2368 (sec21-1, by using genetic methods described in Sherman et al. (1986). which is linked to RFC3), and CH595 (POL30, to identify Suppressors of Cold-Sensitive PCNA Mutations 1619

TABLE 1 S. cerevisiae strains used in this study

Strain Genotypea Source or reference CH526 MATa ade1 ade2 ura1 lys2 tyr1 gal1 his7 cdc2 D. Botstein CH756 MATa ade1 ade2 his7 lys2 tyr1 ura1 cdc8-1 L. Hartwell CH1785 MATa his3 leu2 trp1 ura3 ira2::HIS3 b F. Tamanoi CH2165 MATa leu2-3,112 ura3-52 POL30.LEU2 Amin and Holm (1996) CH2158 MATa leu2-3,112 ura3-52 pol30-101.LEU2 Amin and Holm (1996) CH2180 MAT␣ leu2-3,112 ura3-52 ade2-101 pol30-101.LEU2 RFC1.URA3 This study CH2159 MATa leu2-3,112 ura3-52 pol30-100.LEU2 Amin and Holm (1996) CH2179 MAT␣ leu2-3,112 ura3-52 ade2-101 trp1⌬1 pol30-100.LEU2 RFC1.URA3 This study CH2161 MATa leu2-3,112 ura3-52 pol30-104.LEU2 Amin and Holm (1996) CH2181 MAT␣ leu2-3,112 ura3-52 ade2-101 pol30-104.LEU2 RFC1.URA3 This study CH2162 MATa leu2-3,112 ura3-52 pol30-102.LEU2 Amin and Holm (1996) CH2170 MATa leu2-3,112 ura3-52 pol30-106.LEU2 Amin and Holm (1996) CH2183 MAT␣ leu2-3,112 ura3-52 ade2-101 pol30-106.LEU2 RFC1.URA3 This study CH2171 MATa leu2-3,112 ura3-52 pol30-108.LEU2 Amin and Holm (1996) CH2184 MAT␣ leu2-3,112 ura3-52 ade2-101 pol30-108.LEU2 RFC1.URA3 This study CH2369 MATa leu2-3,112 ura3-52 pol30-114.LEU2 B. Merrill and C. Holm CH2252 MATa leu2-3,112 ura3-52 pol30-104.LEU2 [rho0] Amin and Holm (1996) CH2253 MATa leu2-3,112 ura3-52 POL30.LEU2 [rho0] Amin and Holm (1996) CH2431 MATa leu2-3,112 ura3-52 pol30-104.LEU2 RFC3-3 [rho0] This study CH2234 MATa trp1-289 ura3-1, 2 ade2-101 gal2 can1 pol2-11 J. Campbell CH2237 MATa leu2-3,112 ura3-52 ade2-101 lys2 RFC1.URA3 This study CH2384 MATa leu2 ura3 his3 suc2⌬9 sec21-1 S. Emr CH2385 MAT␣ leu2-3,112 ura3-52 his3 sec21-1 pol30-100.LEU2 RFC2-10 This study CH2386 MATa leu2-3,112 ura3-52 pol30-104.LEU2 RFC3-1 This study CH2387 MATa leu2-3,112 ura3-52 ade2 pol30-104.LEU2 RFC3-2 This study CH2388 MATa leu2-3,112 ura3-52 pol30-104.LEU2 RFC3-1 This study CH2389, CH2390 MATa leu2-3,112 ura3-52 pol30-104.LEU2 RFC3-2 This study CH2391, CH2392 MATa leu2-3,112 ura3-52 pol30-104.LEU2 RFC3-3 This study CH2401 MATa leu2-3,112 ura3-52 trp1-289 prb1-1122 prc1-407 pep4-3 Gerik et al. (1997) CH2404 MATa leu2-3,112 ura3-52 trp1-289 prb1-1122 prc1-407 pep4-3 [pCH1655 Gerik et al. (1997) (pGAL1-GAL4 LEU2)] and [pCH1656 (pGAL1-10 RFC1 RFC2 RFC3 RFC4 RFC5 TRP1)] CH2405, CH2406 MATa leu2-3,112 ura3-52 pol30-100.LEU2 RFC1-19 This study CH2407, CH2408 MATa leu2-3,112 ura3-52 pol30-100.LEU2 RFC2-10 This study CH2409, CH2410 MATa leu2-3,112 ura3-52 pol30-100.LEU2 RFC3-3 This study CH2411 MATa leu2-3,112 ura3-52 pol30-100,120.LEU2 This study CH2412, CH2413, MATa leu2-3,112 ura3-52 pol30-104,121.LEU2 This study CH2414 CH2415, CH2416 MATa leu2-3,112 ura3-52 pol30-104,122.LEU2 This study CH2417 MATa leu2-3,112 ura3-52 pol30-108.LEU2 RFC3-3 This study CH2418, CH2419, MATa leu2-3,112 ura3-52 pol30-108,121.LEU2 This study CH2420 CH2421 MATa leu2-3,112 ura3-52 pol30-108,123.LEU2 This study CH2422 MATa leu2-3,112 ura3-52 pol30-100,124.LEU2 This study CH2423 MATa leu2-3,112 ura3-52 pol30-100,125.LEU2 This study CH2586 MATa leu2-3,112 ura3-52 trp1-289 prb1-1122 prc1-407 pep4-3 [pCH1667 Gerik et al. (1997) (pGAL1-10 RFC1 RFC2 RFC4 RFC5 TRP1)] and [pCH1669 (pGAL1-10 RFC3-3 LEU2)] a The URA3 or LEU2 genes in these strains are markers for a described gene but do not disrupt the gene. b The HIS3 gene in this strain disrupts the IRA2 gene. suppressor mutations in pol30 or RFC5). In a cross between sors of the pol30-100 mutation were identiﬁed as RFC1-19 in strain CH2368 (sec21-1) and CH2386 (pol30-104 SOP1sup), we a cross between strains CH2237 (RFC1.URA3) and CH2405 found that of the 29 spores that contained an unsuppressed (pol30-100 RFC1-19) or CH2406 (pol30-100 RFC1-19). The re- cold-sensitive pol30-104 mutation, 26 of the spores contained maining extragenic suppressors of pol30-100 and the single the sec21-1 mutation. This result indicated that the SOP1 gene extragenic suppressor of pol30-108 were identiﬁed as RFC2-10 is linked to the SEC21 gene, which is 6 kb away from RFC3 or RFC3-3 by directly sequencing the genomic copy of these on chromosome XIV. Similarly, two of the extragenic suppres- genes. 1620 N. S. Amin, K. M. Tuffo and C. Holm

Flow cytometry: The extent of suppression of the cold-sensi- strain CH2404. To purify RFCsup, we constructed strain tive defect of pol30 mutant cells was examined by flow cytome- CH2586 by transforming strain CH2401 with plasmids try. Briefly, rho0 strains CH2253 (POL30), CH2252 (pol30- pCH1669 (RFC3-3; created by replacing RFC3 in pBL413 with 104), and CH2431 (pol30-104 RFC3-3) were grown to log phase a fragment containing the RFC3-3 mutation from plasmid in YEPD at 35Њ. The asynchronously growing cultures were pCH1663 using the NcoI and StuI restriction enzymes; plasmid then divided into two portions; one was incubated at 35Њ for pBL413 was obtained from Peter Burgers) and pCH1667 3 hr, and the other was incubated at 14Њ for 24 hr. Samples were (pBL425 containing RFC1, RFC2, RFC4, and RFC5 under the collected, sonicated, and then prepared for flow cytometry by control of GAL1-10 UAS; Gerik et al. 1997). The yeast strains staining with propidium iodide (Hutter and Eipel 1979). were grown in selective media containing 3% glycerol, 2% For each sample, 10,000 cells were counted to assess the DNA lactate, and 0.1% glucose for 2 days, followed by a 3-hr incuba- content by a Becton Dickinson (San Jose, CA) cell fluores- tion in rich media containing 2% lactate, 0.2% glucose, and cence cell sorter. 2% galactose. Cell noodles were made with 600 g of cells using Allele-restricted suppression of pol30 alleles: To determine liquid nitrogen, and they were ground in a motorized grinder whether suppression was allele-restricted, each SOP allele was under liquid nitrogen. The ground cells were resuspended in tested to determine whether it could suppress various CsϪ and buffer A (25 mm Tris-HCl pH 7.5, 10% glycerol, 1 mm DTT, 1 Mmss mutant alleles of pol30. For this purpose, suppressor mm EDTA, 0.01% Nonidet P-40, 0.1 mm phenylmethylsulfonyl strains CH2405 (pol30-100 RFC1-19), CH2385 (pol30-100 RFC2- fluoride, 5 ␮m pepstatin A, and 5 ␮m leupeptin) containing 1), CH2386 (pol30-104 RFC3-1), or CH2387 (pol30-104 RFC3- 500 mm NaCl, and they were pelleted. The extracts were frac- 2) were crossed with strains CH2165 (POL30), CH2158 (pol30- tionated first by using ammonium sulfate, which was added 100), CH2179 (pol30-100), CH2159 (pol30-101), CH2180 to 5% saturation, followed by fractionation with 65% satura- (pol30-101), CH2162 (pol30-102), CH2161(pol30-104), tion of ammonium sulfate. The pellet was resuspended in CH2181 (pol30-104), CH2170 (pol30-106), CH2183 (pol30- buffer A containing 200 mm NaCl to obtain a protein concen- 106), CH2171 (pol30-108), CH2184 (pol30-108), or CH2369 tration of 5–10 mg/ml. The solution was dialyzed overnight (pol30-114). Tetrad analysis was performed and the spores with buffer A and the extracts were subjected to a phosphocel- were tested for sensitivity to cold (20Њ or 14Њ) and sensitivity lulose P11 column. The column was equilibrated and washed to different concentrations of MMS (0.01, 0.015, or 0.02%). with buffer A containing 200 mm NaCl before elution with To test whether the RFC3-3 mutation suppresses pol30 alleles buffer A containing 400 mm NaCl followed by buffer A con- other than pol30-100, pol30-104,orpol30-108, the RFC3 gene taining 800 mm NaCl. The fractions eluted with buffer A con- or the RFC3-3 gene was amplified from the genome of strain taining 800 mm NaCl were collected and assayed for protein CH2171 (RFC3) or CH2409 (RFC3-3), respectively. The 2.3- amount using the Bradford assay. The peak fractions were kb PCR fragments containing either RFC3 or RFC3-3 were pooled (615 mg protein) and chromatographed further using digested with SspI and ligated into a SmaI-digested vector a hydroxyapatite column, a single-stranded DNA column, and pCH1099 (CEN6 ARS1 URA3). The resulting plasmids a Q Sepharose column as described in Lee et al. (1991a). We pCH1662 (RFC3 URA3) and pCH1663 (RFC3-3 URA3) were monitored the purification of wild type and mutant RFC using each transformed into strains CH2165 (POL30), CH2158 the DNA synthesis assay described below. We obtained 6 mg (pol30-100), CH2159 (pol30-101), CH2161 (pol30-104), of protein after the final step. CH2162 (pol30-102), CH2170 (pol30-106), CH2171 (pol30- PCNA and DNA polymerase ␦ purification: To purify wild- 108), or CH2369 (pol30-114). The transformants were tested type and mutant PCNA proteins we followed the protocol for sensitivity to cold (20Њ or 14Њ) and sensitivity to different outlined in Fien and Stillman (1992). The hexahistidine- concentrations of MMS (0.01, 0.015, or 0.02%). tagged (his-tagged) expression plasmid containing wild-type Genomic sequencing: To identify a nucleotide change(s) POL30, plasmid pCH1695, was obtained from Mike McAlear between the original pol30, RFC1, RFC2,orRFC3 alleles and [pMM115, which has the POL30 gene cloned into a pRSETA their suppressing counterparts, the suppressor genes were se- vector from Invitrogen (San Diego); Beckwith et al. (1998)]. quenced using a PCR-based genomic sequencing technique To purify his-tagged Pol30-104p, we replaced a ClaI-HindIII (Promega, Madison, WI). Briefly, the genomic suppressor fragment in plasmid pCH1695 with a ClaI-HindIII fragment gene was amplified using genomic DNA isolated (Sherman containing the pol30-104 mutation from plasmid pCH1598 et al. 1986) from each suppressor strain by PCR using Taq (Amin and Holm 1996) to produce plasmid pCH1700. To polymerase (AmpliTaq; Perkin Elmer, Norwalk, CT). The purify S. cerevisiae DNA polymerase ␦ (pol ␦) we used the wild-type POL30, RFC1, RFC2,orRFC3 genes were also ampli- protocols outlined in Bauer et al. (1988) and Zuo et al. (1997). fied from unsuppressed pol30 strains, and they were used as Briefly, we obtained a protease-deficient yeast strain RDKY- controls in the entire sequencing process. In 20 sequencing 1293 from Richard Kolodner, and we prepared cell extracts to runs, we never observed a sequencing gel that showed an purify native pol ␦. The extracts were subjected to ammonium apparent mutation in the wild-type sequence. For the PCR sulfate fractionation and column chromatography using phos- amplification of genes, multiple independent PCR reactions phocellulose P11, Q Sepharose, SP Sepharose, a single- were pooled together to prevent an accumulation of single stranded DNA cellulose, and hydroxyapatite columns in a “jackpot” mutations caused from errors that could be intro- sequential manner. The fractions obtained were assayed for duced by the Taq polymerase. The amplified wild-type and DNA synthesis activity (described below) during the purifica- suppressor genes were then sequenced using the cycle se- tion process. quencing kit from Promega. DNA synthesis assays: To carry out the DNA synthesis assay Purification of wild-type and mutant RFC: To purify wild- we prepared an end-labeled singly primed DNA template using type and mutant RFC complexes we used a procedure that ⌽X174 viral DNA as template (5386 nucleotides; New England was similar to one previously described in Lee et al. (1991a). Biolabs, Beverly, MA) and a 30-mer primer that anneals to it To purify wild-type RFC, protease-deficient strain CH2401 from nucleotide 5127–5156 as described previously (Lee et al. (BJ2168; Gerik et al. 1997) was transformed with plasmid 1991a). The DNA synthesis reaction (30 ␮l) was carried out pCH1656 (pBL420 containing wild-type RFC1, RFC2, RFC3, using 250 fmol of pol ␦, 240 fmol of wild-type RFC or 50 fmol RFC4, and RFC5 under the control of GAL1-10 UAS; Gerik et of RFCsup protein, 1000 fmol of wild-type or mutant PCNA al. 1997) and plasmid pCH1655 (pMTL4 containing GAL4 protein, 300 ng of Escherichia coli single-strand binding protein under the control of GAL1 UAS; Gerik et al. 1997) to produce (Pharmacia, Piscataway, NJ), and 300 fmol of primer/tem- Suppressors of Cold-Sensitive PCNA Mutations 1621

plate. In addition, the reaction contained 100 ␮m dNTP, 2 could compensate for the defects caused by the original ␮ mm ATP, 7 mm MgCl2,1 g BSA, 0.5 mm DTT, and 40 mm mutation, and we recovered a total of 12 such intragenic Tris pH 7.8. The reaction was incubated at 37Њ for 30 min and then stopped with 1 ␮l of 0.1 m EDTA and 2 ␮lof6ϫ suppressor mutations. To demonstrate that each of the dye (0.25% bromophenol blue and 30% glycerol). The reac- suppressor mutations was linked to the pol30 gene, we tions were loaded onto a 1% agarose gel containing 50 mm crossed the pol30 sup strains with a wild-type POL30 strain NaOH and 3 mm EDTA. The gel was run for 16 hr at 35 V, (CH2237). We observed no CsϪ spores in any of the and it was dried and exposed to film. crosses upon tetrad analysis (at least seven tetrads were The DNA synthesis assay conditions used during the purification process were similar to the assay described above with analyzed for each strain). Using genomic sequencing the exception that 33.3 ␮m of [3H]dCTP and unlabeled ⌽X174 we confirmed that all the suppressor mutations that primer/template were used during the reaction instead of were linked to the pol30 locus were indeed within the 32P-end-labeled ⌽X174 primer/template. The reactions were pol30 gene. Specifically, we observed a single second incubated for 30 min at 37Њ or 25Њ. Next, 10 ␮l of 10 mg/mL pol30 mutation in every intragenic suppressor strain; no salmon sperm DNA, 100 ␮lof0.1m sodium pyrophosphate, and 5 ml of 5% TCA were added to each reaction. The reac- back mutations restoring the wild-type POL30 sequence tions were incubated on ice for 10 min, after which they were were observed. The amino acid changes identified in poured over 25-mm glass fiber filters (ENZO Diagnostics) to the case of the 12 intragenic suppressor strains affect filter out the unincorporated 3H nucleotides. The filters were five amino acids of PCNA: pol30-120 (D41N), pol30-121 washed once with 5% TCA, once with 1% TCA, and then with (D71Y), pol30-122 (D41G), pol30-123 (L205S), pol30-124 ethanol. The filters were dried and then counted using a scintillation counter. (G218C), and pol30-125 (S219P). It is striking that amino acid changes affecting aspartic acid 41 are seen in suppressors of both pol30-100 and pol30-104. Simi- RESULTS larly, the same amino acid change affecting aspartic acid 71 was observed in a pol30-104 suppressor and a pol30- We screened for proteins that interact functionally 108 suppressor. Mapping the pol30 sup mutations on the with yeast PCNA using the powerful genetic technique crystal structure of PCNA revealed that only one of the of pseudoreversion, or second-site suppression. This ap- suppressor mutations altered an amino acid close to the proach has been used successfully to identify many gene one changed by the original pol30 mutation (Figure products, such as ␥-tubulin, which interacts with ␤-tubu- 1). One obvious possibility is that all of the suppressor lin, and the actin-binding protein fimbrin (Adams and mutations alter the 3-D structure in such a way as to Botstein 1989; Oakley and Oakley 1989). We ex- compensate for the original alteration. pected to recover intragenic suppressors affecting the Defects in pol30-104 (A251V) and pol30-108 (A251T) pol30 gene itself using this method. More important, if mutants are suppressed by dominant mutations in RFC3: an interaction with another protein is affected by the To learn about proteins that interact with PCNA, we original pol30 mutation, we hoped to recover extragenic analyzed the extragenic suppressors of pol30 mutations. suppressors that were dominant and allele-restricted for Because alanine 252 of human PCNA is known to inter- suppression (Adams and Botstein 1989; McAlear et act directly with the C-terminal region of p21, we first al. 1994; Sandrock et al. 1997). isolated second-site suppressors of a CsϪ mutant of yeast We selected spontaneously arising mutations that can PCNA (pol30-104) that affects the equivalent residue, compensate for the DNA replication and DNA repair alanine 251. Genetic analysis of five revertant strains defects of PCNA mutants. To isolate suppressors of CsϪ showed not only that suppression of the mutant pheno- pol30 mutants, we incubated strains CH2159 (pol30- types was due to extragenic mutations affecting single 100), CH2161 (pol30-104), and CH2171 (pol30-108)at genes, but that all five of the suppressors mapped to the semipermissive (20Њ) or nonpermissive temperature the same locus (SOP1). Furthermore, diploid strains (14Њ). Spontaneously arising Csϩ colonies (revertants) that are homozygous for pol30-104 and heterozygous for were selected and analyzed genetically. All of the sup- SOP1sup exhibit dominant suppression in all cases. Thus, pressor mutations suppress the CsϪ phenotype of the even in the presence of the wild-type copy of SOP1, the original pol30 mutations equally well at both tempera- SOP1sup allele is able to restore function to the mutant tures. Apart from the suppression phenotype, the sup- PCNA. Because all of the suppressors confer dominant pressors conferred no new phenotypes, such as heat- suppression and affect the same gene product, our re- sensitivity or sensitivity to the DNA synthesis inhibitor sults suggested that the SOP1 gene product is required hydroxyurea. Genetic analysis revealed that suppression for proper function of PCNA. was linked to a single gene [suppressor of pol30 (SOP)1, To determine the extent of suppression of pol30-104 2, 3, etc.] in each of the strains. Both intragenic and by SOP1sup, flow cytometry was used to examine the DNA extragenic suppressors were obtained for each of the content of asynchronously dividing cultures of POL30, three pol30 alleles. pol30-104, and pol30-104 SOP1sup strains at the permissive Several intragenic mutations suppress the defects (35Њ) and nonpermissive temperatures (14Њ). While the caused by the original pol30 mutations: Each pol30 mu- DNA profiles of all strains were similar at the permissive tant gave rise to at least one new pol30 mutation that temperature, the DNA profiles at the restrictive temper- 1622 N. S. Amin, K. M. Tuffo and C. Holm

Figure 2.—DNA content of POL30, pol30, and pol30 SOP1 strains. Early log phase cultures of the rho0 strains CH2253 (POL30), CH2252 (pol30-104), and CH2431 (pol30-104 SOP1-3) were shifted to the permissive temperature (35Њ) for 3 hr or the restrictive temperature (14Њ) for 24 hr. Propidium iodide staining and flow cytometry were subsequently used to monitor the DNA content of the cells (see materials and methods). The x-axis represents the relative DNA content and the y-axis Figure 1.—Locations of amino acids altered in strains car- represents cell number. rying intragenic suppressors of pol30 mutations. The structure of a PCNA monomer is shown using the Rasmol program. A second mutation within the pol30 gene itself was found in 12 of 24 pseudorevertant strains that were isolated using three whether SOP1 encodes a protein that has been pre- cold-sensitive pol30 mutants. The original amino acids viously suggested from biochemical studies to interact changed in cold-sensitive pol30 mutants [pol30-100 (K253E), with PCNA. Obvious candidates include both the pro- pol30-104 (A251V), and pol30-108 (A251T)] are shown in teins of the RFC complex that loads PCNA onto the green in the yeast PCNA monomer structure (Krishna et al. DNA and the genes encoding the catalytic subunits of 1994). The residues affected in the 12 intragenic suppressor ε strains map to five different amino acids, which are shown in DNA polymerases ␦ and . Thus, we performed crosses red [D41N (pol30-120); D41G (pol30-122); D71Y (pol30-121); to test whether the SOP1 gene was RFC1, RFC2, RFC3, L205S (pol30-123); G218C (pol30-124); and S219P (pol30- RFC4, RFC5, CDC2 (encodes the catalytic subunit of 125)]. DNA polymerase ␦), or POL2 (encodes the catalytic subunit of DNA polymerase ε). Tetrad analysis revealed that the SOP1 gene is linked to the SEC21 locus, sug- ature differed significantly (Figure 2). Whereas the gesting that the neighboring RFC3 gene was a likely POL30 culture contains cells in both the G1 and G2 candidate. We confirmed that SOP1 is indeed RFC3 by phases of the cell cycle, the majority of cells in the pol30- genomic sequencing (Table 2) and by showing that an 104 strain are arrested in the G2 phase of the cell cycle. RFC3 suppressor allele (RFC3-3) is sufficient to suppress In contrast, the suppressor strains show an intermediate a pol30-104 mutation when carried on a plasmid (data phenotype. Because we have previously shown that the not shown). This in vivo result gives biological support G2 arrest of pol30-104 cells is due to a defect in progres- to biochemical results that show that both the RFC com- sion through S phase (Amin and Holm 1996), these plex (Tsurimoto and Stillman 1991a; Gerik et al. results suggest that the DNA replication defect of the 1997) and RFC3p in particular interact physically with pol30-104 mutation is partially suppressed in the pol30- PCNA (Mossi et al. 1997). 104 SOP1sup double mutant strain. The generality of suppression of pol30 defects by RFC3 To determine the identity of the SOP1 gene, we tested was demonstrated when a mutation in RFC3 was isolated Suppressors of Cold-Sensitive PCNA Mutations 1623

TABLE 2 Sequence changes in the extragenic suppressors of pol30 alleles

No. of Suppressor Nucleotide Amino acid pol30 allele suppressor strains genea change change pol30-100 2 RFC1-19 G1189C D397H (K253E) 2b RFC2-10 C236A T79K 2b RFC3-3 G234A M78I pol30-104 1 RFC3-1 A229T N77Y (A251V) 2 RFC3-2 A232G M78V 2 RFC3-3 G234A M78I pol30-108 1 RFC3-3 G234A M78I (A251T) a Uppercase letters are used to represent all the RFC suppressor genes because they are dominant for suppression of pol30 mutant phenotypes. b Each of these pairs of strains came from the same original pol30-100 mutant cultures. Therefore, they contain mutations that may be identical by descent. as a suppressor of a second allele of pol30, pol30-108. three specific RFC members, RFC1p, RFC2p, and Like pol30-104, pol30-108 causes an amino acid change RFC3p that are important for a functional interaction in alanine 251 of yeast PCNA, but with pol30-108 the with the interdomain region of yeast PCNA. change is to a threonine instead of valine. Only one Allele-restricted suppression of different pol30 alleles pseudorevertant strain of pol30-108 contained an ex- by RFC3 suppressor alleles: To determine whether the tragenic suppressor. Genetic analysis revealed that this suppressor mutations suppress specific defects in PCNA, suppressor maps to a single gene that confers dominant we next examined the allele specificity of suppression suppression. Using genomic sequencing we determined of pol30 alleles by RFC mutations. We used tetrad analysis once again that the suppressor mutation was in the RFC3 or plasmid transformation to determine whether any of gene (Table 2). This result confirms that the RFC3 gene the RFC suppressors can suppress a collection of pol30 product is important for the functional interaction with alleles that have previously been isolated (Table 3). All the interdomain region of PCNA. of the RFC suppressor mutations suppress the pheno- The pol30-100 (K253E) mutation is suppressed by mu- types of some, but not all, CsϪ pol30 alleles. For example, tations affecting not only RFC3 but also RFC1 and RFC2: we observed that the RFC3-1 mutation, which was recov- To determine whether proteins other than RFC3p ered as a suppressor of pol30-104 (A251V), fails to sup- might also interact with the interdomain region of press the CsϪ phenotype of the pol30-101 mutation PCNA, we chose a different CsϪ mutation to isolate (R44G), which is located farthest away from the pol30- additional second-site suppressors. The pol30-100 muta- 104 mutation. In addition, the CsϪ pol30-106 mutation, tion causes an amino acid change from lysine 253 to which is suppressed by all three RFC3 suppressors, is glutamic acid, and it can be distinguished from the not suppressed by the RFC1 or RFC2 mutations. Suppres- pol30-104 and pol30-108 mutants by its more severe DNA sion of the Mmss phenotype was even more specific; replication defect (B. J. Merrill and C. Holm, unpub- sensitivity to MMS was usually suppressed in at most two lished data). Six pseudorevertants of the pol30-100 mu- of the pol30 alleles. The observation that the RFC3-2 tant were analyzed genetically to identify the gene prod- mutation suppresses the phenotype of pol30-104 and ucts affecting suppression. Genetic mapping and not pol30-108 is particularly striking because both of genomic sequencing (Table 2) revealed that the six these pol30 mutations affect alanine 251, although with extragenic suppressors of pol30-100 affect three differ- different amino acid changes. The differences in sup- ent genes. Two of the six suppressor genes mapped to pression of pol30 alleles by the RFC suppressors suggest the RFC3 locus in this third independent screen. In that the requirement of PCNA in MMS-induced DNA addition, two independently derived pseudorevertant repair is likely to involve a different mechanism of action strains proved to carry dominant suppressor mutations or different levels of activity compared to its role in in the RFC1 gene, which encodes the large subunit of DNA replication. Overall, these results demonstrate that replication factor C. The remaining two pseudore- suppression by RFC mutations is allele-restricted. Be- vertant strains carry dominant mutations affecting yet cause dominant allele-restricted suppression suggests another member of the RFC gene family, the RFC2 gene. an interaction between proteins (Adams and Botstein In summary, although the importance of the RFC com- 1989; Sandrock et al. 1997), these results are consistent plex as the loader of the PCNA clamp has been shown with a functional interaction between RFC1p, RFC2p, biochemically, using genetic studies we have identified and RFC3p and PCNA. 1624 N. S. Amin, K. M. Tuffo and C. Holm

TABLE 3 Suppression of CsϪ and/or MmsϪ phenotypes of pol30 alleles by RFC sup alleles

pol30 allelea RFC1-19 RFC2-10 RFC3-1 RFC3-2 RFC3-3 b pol30-104 (A251V) CsϪ MmsϪ Csϩ Mmsϩ Csϩ Mmsϩ Csϩ Mmsϩ Csϩ MmsϪ pol30-100 (K253E) Csϩ Mmsϩ Csϩ Mmsϩ Csϩ/Ϫ MmsϪ Csϩ MmsϪ Csϩ Mmsϩ/Ϫ pol30-101 (R44G) Csϩ Mmsϩ/Ϫ Csϩ Mmsϩ CsϪ MmsϪ Csϩ MmsϪ Csϩ Mmsϩ/Ϫ pol30-106 (P234S) CsϪ MmsϪ CsϪ MmsϪ Csϩ MmsϪ Csϩ MmsϪ Csϩ Mmsϩ/Ϫ pol30-108 (A251T) CsϪ MmsϪ Csϩ Mmsϩ Csϩ Mmsϩ Csϩ MmsϪ Csϩ Mmsϩ pol30-102 (G244R)c ND ND MmsϪ MmsϪ MmsϪ pol30-114 (K13E)c ND ND MmsϪ MmsϪ MmsϪ The symbols ϩ, ϩ/Ϫ, and Ϫ indicate complete, partial, and lack of suppression, respectively, of the cold- sensitive (CsϪ) or MMS-sensitive (MmsϪ) phenotype of pol30 alleles by RFC suppressor alleles. a The pol30 alleles used in this experiment are described in Amin and Holm (1996). b A plasmid-borne allele of RFC3-3 was used to assay suppression of pol30 mutant phenotypes, whereas suppression of pol30 mutant defects by other RFC sup alleles was determined by tetrad analysis. c These pol30 alleles are MmsϪ and not CsϪ; therefore, only results for Mms-sensitivity are reported. Suppres- sion of these alleles by RFC1-19 or RFC2-10 was not determined (ND).

Genomic sequencing of the RFC suppressor genes acid sequence of the respective genes revealed that all reveals a common region that is important for suppres- of the extragenic suppressors have amino acid changes sion of pol30 mutations: To evaluate the conservation in the region of RFC box IV (Figure 3), which is one of the regions of RFC1p, RFC2p, and RFC3p that are of eight regions conserved among the RFC genes (Cull- important for interaction with PCNA in vivo,wese- man et al. 1995). Although the function of RFC box IV quenced the genomic copy of RFC1, RFC2, and RFC3 in is not apparent from its sequence, RFC boxes III and the pseudorevertant strains (Table 2). We saw a striking V have sequence similarity to the ATP-binding region clustering of amino acid changes in asparagine 77 or of known ATPases. Our results suggest that RFC box IV methionine 78 in RFC3p (8 of 12 extragenic suppres- may play an additional role in functionally interacting sors; Table 2 and Figure 3). Furthermore, regardless of with PCNA. which pol30 mutation was used in the pseudoreversion RFC3 suppressor mutations suppress the in vitro DNA screen, we obtained the same amino acid change in synthesis defect in Pol30-104p: To begin to understand RFC3p from methionine 78 to isoleucine in ﬁve differ- the defect in pol30 mutants that is suppressed by RFC ent suppressor strains (Table 2). Our results suggest mutations, we examined the characteristics of the mu- that asparagine 77 and methionine 78 of RFC3p are tant proteins biochemically. Because pol30 mutants syn- critical for RFC3p function because they either affect thesize DNA very slowly at the restrictive temperature, protein folding or they are important points of contact it seemed likely that the biochemical defect in the pol30 with PCNA; this methionine is present in RFC3p of both mutant proteins would be a processivity defect. To deter- yeast and humans (Cullman et al. 1995). Mapping the mine whether Pol30-104p causes DNA synthesis defects RFC1, RFC2, and RFC3 mutations onto the linear amino in vitro, we used a singly primed ⌽X174 DNA template

Figure 3.—RFC suppressor mutations map near RFC box IV. Although RFC1p is the only RFC protein to have RFC box I (DNA ligase homology box), all RFC proteins share the conserved RFC boxes II–VIII (Cullman et al. 1995). Map- ping the RFC1, RFC2, and RFC3 suppressor mutations (arrows) onto the linear sequence of the respective genes reveals a striking clustering of the amino acid changes in the region of RFC box IV (hatched box). Although the function of RFC box IV is not apparent from its sequence, the clustering of suppressor mutations nearby suggests that it may be important for interactions with PCNA. (This is modeled after Figure 8 in Cullman et al. 1995.) Suppressors of Cold-Sensitive PCNA Mutations 1625

combination of proteins pauses less on the template DNA, and it is able to promote the synthesis of full- length ⌽X174 DNA even at 25Њ (Figure 4, lanes 11 and 12). If the lack of full-length ⌽X174 DNA synthesis accu- rately reflects the in vivo defect of the pol30-104 mutant, then this defect should be suppressed in vitro by the addition of the mutant RFCsup complex, because RFC sup suppresses the in vivo defect. Indeed, in lanes 13–16 of Figure 4, it is clear that suppressor RFC, which contains the RFC3-3 protein, alleviates the DNA synthesis defect; even though there is fivefold less RFCsup protein than wild-type RFC in this experiment, the Pol30-104 protein promotes the efficient production of full-length ⌽X174 DNA (Figure 4, lanes 13 and 14; similar results were obtained in reactions in which the same amount of RFCsup or wild-type RFC was added). To determine whether the in vitro phenotype also holds true for another PCNA mutant whose CsϪ phenotype is also suppressed by RFC3-3, we examined in vitro DNA synthesis Figure 4.—Effect of wild-type and mutant PCNA and RFC proteins in pol ␦-dependent DNA synthesis. Combinations of with the Pol30-106 protein in reactions with wild-type mutant and wild-type RFC and PCNA were assayed for their or suppressor RFC. Consistent with the in vivo results ability to promote pol␦-dependent DNA synthesis at 37Њ (lanes (Table 3), the DNA synthesis defect in Pol30-106p is 1, 2, 5, 6, 9, 10, 13, and 14) or 25Њ (lanes 3, 4, 7, 8, 11, 12, also suppressed by an RFCsup complex containing RFC3- 15, and 16) in 30 min. Each reaction contained identical 3p (data not shown). amounts of singly primed ⌽X174 DNA, pol␦, and E. coli single- strand binding protein (see materials and methods). In addition, each reaction contained either wild-type (wt Pol30) DISCUSSION or mutant (Pol30-104) PCNA, and either wild-type (wt RFC) or suppressor (sup RFC) replication factor C, as indicated We have identified spontaneously arising mutations at the top. Full-length ⌽X174 is 5.4 kb long. DNA product in the RFC1, RFC2, and RFC3 genes that suppress the formation was assayed by using alkaline agarose gel electro- Ϫ phoresis followed by autoradiography. Details of the reaction DNA replication and DNA repair defects of Cs pol30 conditions are described in materials and methods. mutants. All of the suppressor mutations confer a dominant suppression phenotype and show allele-restricted suppression of pol30 alleles. To elucidate the regions of to examine the ability of wild-type and mutant PCNA RFC1, RFC2, and RFC3 that are important for suppres- and RFC proteins to stimulate Pol␦-dependent DNA sion of pol30 mutant defects, we performed genomic synthesis. Although wild-type RFC with wild-type PCNA sequencing. Our results suggest that the region around promotes a significant amount of DNA synthesis of full- RFC box IV, which is conserved in all of the RFC genes, is length ⌽X174 DNA (5386 nucleotides) within 30 min important for a functional interaction of RFC1p, RFC2p, at 37Њ (Figure 4, lanes 1 and 2), wild-type RFC with and RFC3p with the interdomain region of PCNA. In the Pol30-104 protein promotes very little full-length vitro analysis of the wild-type and mutant RFC and PCNA synthesis under the same conditions (Figure 4, lanes 5 proteins reveals that mutant PCNA proteins during pol and 6). Similar results were obtained when the incuba- ␦-dependent DNA synthesis are defective in enhancing tion times were reduced to 3, 5, or 10 min (data not elongation and that this defect is suppressed by a mutant shown). These results are consistent with a defect in RFC complex containing suppressor RFC3-3p. processivity in the Pol30-104 protein. Our second-site suppression screens were successful It is interesting to note that the production of full- in identifying specific proteins that interact with PCNA length product is dramatically affected by the tempera- in vivo. Many biochemical studies have previously shown ture of the assay in all samples. For example, at 25Њ even that PCNA can interact with a large number of proteins, DNA polymerase complexes containing wild-type RFC such as the RFC complex (Tsurimoto and Stillman and PCNA proteins appear to stall when the DNA prod- 1991a; Gerik et al. 1997), DNA polymerase ␦ (Bauer uct reaches one-fifth of its full length (Figure 4, lanes and Burgers 1988; Zhang et al. 1995), FEN-1 (Li et 3 and 4). One explanation for this phenomenon is that al. 1995), Msh2/Msh3 (Johnson et al. 1996), Gadd45 the secondary structure of the template DNA may be (Chen et al. 1995), a DNA methylation protein (Chuang substantially more stable at the lower temperature. This et al. 1997), and p21WAF1/CIP1 (Xiong et al. 1992, 1993; problem is circumvented by the presence of the RFCsup Zhang et al. 1993; Flores-Rozas et al. 1994; Waga et complex in reactions containing wild-type PCNA; this al. 1994). Our studies suggest that out of these possible 1626 N. S. Amin, K. M. Tuffo and C. Holm interactions, only the RFC interactions can compensate physical interaction between two proteins (Jarvik and for the cold sensitivity caused by pol30 mutations in the Botstein 1973), or it can result from a more general interdomain region of yeast PCNA. It may be the case physical or functional interaction (Sandrock et al. that our pol30 alleles are cold sensitive because of the 1997). In general these two forms of suppression can lack of a specific interaction of PCNA with the RFC be distinguished as follows. If suppression derives from complex, which could result in a DNA synthesis defect. the restoration of a specific physical interaction, then Of course, conclusions about the specificity of this inter- suppression is generally strictly allele specific, and interaction are somewhat limited because our screens were action between the suppressor proteins and a wild-type not fully saturated; although RFC1-19 and RFC2-10 were protein is frequently compromised. If suppression de- identified as suppressors of pol30-100, these mutations rives from the creation of a new functional or physical were not recovered from pseudoreversion screens with interaction, then suppression is more general (allele- the pol30-104 and pol30-108 mutants. Nonetheless, it is restricted), and the suppressor protein may exhibit en- striking that among 12 extragenic suppressors, all 12 hanced binding to the wild-type protein. As an example affected subunits of the RFC complex. This result sug- of this latter type of suppression, the phenotype of many gests that the most critical interaction with the interdo- actin mutant alleles is suppressed by any one of a num- main region of PCNA is with subunits of the RFC com- ber of mutations in the SAC6 gene, which encodes an plex. actin-binding protein (Adams and Botstein 1989; Although it is clear that RFC requires five individual Sandrock et al. 1997). In this example, the mechanism subunits to function in vitro in yeast (Gerik et al. 1997), of suppression by the mutant Sac6 proteins is the cre- the in vivo function of each of the RFC proteins remains ation of a new binding site, which has an increased unclear. One possibility is that each of the RFC proteins ability to bind to both the mutant and wild-type actin performs a unique cellular function. RFC2p and RFC5p, proteins (Sandrock et al. 1997). In many ways, our for example, may perform a checkpoint function (Sugi- results with mutant and wild-type RFC and PCNA pro- moto et al. 1996; Noskov et al. 1998; Shimomura et al. teins are similar to the results with actin and SAC6p. 1998). However, the existence of only one mutant allele We observe a similar allele-restricted suppression of dif- of each gene makes this result difficult to generalize. A ferent pol30 alleles by the RFC suppressors as well as an second and more likely explanation for the essential increased ability of the RFCsup complex to promote the nature of each RFC gene is that the specific structure synthesis of long DNA products in the presence of wild- created by the assembly of all five RFC subunits is abso- type PCNA during pol ␦-dependent DNA synthesis. lutely essential for the function of the whole complex. Thus, the nature of the suppression is consistent with This hypothesis is supported by increasing biochemical a model in which suppressor mutations in RFC1, RFC2, evidence resulting from purification and analysis of the or RFC3 result in altering the overall affinity or function different subunits of human RFC using recombinant of RFC rather than altering only the specific site where baculoviruses (Cai et al. 1997; Podust and Fanning the amino acid is changed. 1997; Uhlmann et al. 1997a,b). However, it is unclear While RFC can act as a clamp loader and unloader how each of the subunits of RFC within a fully formed based on in vitro reactions, it is not clear from previous complex affects the interaction of the complex with studies whether RFC remains attached to the DNA poly- PCNA. merase machinery during processive DNA elongation. Our in vivo observation that mutations in three differ- If RFC exits after loading PCNA onto the DNA, then ent RFC genes can suppress the single PCNA defect our DNA synthesis results with suppressor RFC would caused by the pol30-100 mutation suggests that these require that RFCsup loads mutant or wild-type PCNA three RFC genes must, at least to a certain extent, play onto the DNA in a way that is both different from normal overlapping roles in the cell. One possibility is that all and stable. This idea is consistent with in vitro studies three subunits (and perhaps RFC4p and RFC5p as well) conducted with human RFC containing an N-terminal make contacts with PCNA in the process of loading it deletion in the largest subunit of RFC; these studies onto the DNA. This idea is consistent with the observa- suggest that RFC is no longer required once PCNA is tion that the human homologs of both RFC1p and loaded onto the DNA (Podust et al. 1998). From other RFC3p interact with the C-terminal face of human biochemical data, however, it seems likely that RFCsup PCNA in vitro (Mossi et al. 1997) and that there is amino remains on the DNA with PCNA in a stable manner. acid sequence conservation among RFC proteins (Cull- For example, Waga and Stillman have recently exam- man et al. 1995). Thus, it is possible that changes in each ined the effect of p21 on PCNA loading by RFC, and of the RFC subunits can cause similar overall functional they show that RFC remains bound to PCNA after the changes in the RFC complex. Alternatively, unique loading reaction; while p21 does not inhibit the loading changes in each of the RFC1, RFC2, or RFC3 proteins process, it causes RFC to dissociate from the PCNA- may compensate for the defect in mutant PCNA pro- bound DNA under conditions allowing ATP hydrolysis teins by acquiring an increased binding capability. (Waga and Stillman 1998). 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