Copyright 1999 by the Genetics Society of America 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 (Cs2) 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 speci®cally looked for new mutations that suppress mutations in the equivalent region of yeast PCNA. In independent screens using three different Cs2 mutants, we identi®ed spontaneously arising dominant suppressor mutations in the RFC3 gene. In addition, dominant suppressor mutations were identi®ed 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 ®ve 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 d-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 ef®ciency 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 ®rst loaded onto the DNA known human mismatch repair genes, such as hMSH2, by a complex of ®ve 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 (d 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 identi®ed 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 suf®cient 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 identi®ed the interdo- For ¯ow cytometry experiments, rho0 strains were created by 1 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 mg/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 d 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. 2 Louis) was added to autoclaved YEPD at a ®nal 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 Cs2 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. Speci®cally, both ef®cient 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 308. 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 148 or the semipermissive temperature of 208. The use of two different 1996). Thus, it is striking that the p21 peptide makes temperatures was initially intended to enhance the speci®city 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 Cs2 and Mmss PCNA where our Cs2 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 sensitiv- ity 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 Cs2 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- 2 of the defects of three Cs pol30 mutants by spontane- gation of the Cs1 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 con®rm that the muta- defects of one of the pol30 alleles.