The Large Subunit of Replication Factor C - Genetics

Copyright 6 1996 by the Genetics Society of America

The Large Subunit of Replication Factor C (Rfclp/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces cerevkiae Michael A. McAlear,’ K Michelle Tuff0 and Connie Holm Department of Pharmacology, Division of Cellular and Molecular Medicine, University of California at San Diego, L a Jolla, California 92093-0651 Manuscript received August 3, 1995 Accepted for publication October 2, 1995 ABSTRACT We used genetic and biochemical techniques to characterize thephenotypes associated withmutations affecting the large subunit of replication factor C (Cdc44p or Rfclp) in Saccharomyces cermisim. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrestand initiate neither mitosis nor a newround of DNA synthesis.cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation.After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after theexcision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.

G

ENETIC and biochemical studies have revealed an underlying conservation in the basic enzymology of DNA replication and DNA repair in a variety of prokaryotic and eukaryotic systems (reviewed in CARR and HOEKSTRA1995;STILLMAN 1994). Among the central proteins that are required for these processes are the DNA polymerases and their accessory proteins. Two classes ofDNA polymerase accessoryproteins thathave been identified are factors knownas “sliding DNA clamps” and the “clamp loaders” (O’DONNELL et al. 1993). Thesetypes ofproteins constitute part of a widely conserved mechanism for enhancing the efficiency of DNA replication. The sliding DNA clamp class of proteins includes the p subunit ofDNA polymerase I11 in Eschm’chia coli and proliferating cell nuclear antigen (PCNA) in eukaryotes. Although unrelated in amino acid sequence, these two proteins share striking functional and structural similarities (KONG et al. 1992; KRISHNA et al. 1994). Ineach case, the proteinsassemble as multimers to form doughnut-shaped rings that can encircle the DNA double helix, tethering theDNA polymerase to the DNA template. Central players in DNA replication in vitro are the clamp loaders, which mayplay an important role in switching DNA synthesis from DNA polymerase a to Corresponding authur: Connie Holm, Department of Pharmacology, Division of Cellular and Molecular Medicine, University of California at San Diego, Mail Code 0651, 9500 Gilman Dr., La Jolla, CA 920930651. E-mail: [email protected] ‘Present address: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459.

Genetics 142 65-78 (January, 1996)

DNA polymerase 6 (TSURIMOTO et al. 1990; EKI et al. 1992). Clamp loaders serve to open the ring-like sliding clamps and assemble them around the double helix (O’DONNELL et al. 1993).Some examples of clamp loaders include the gp44/62 proteins from bacteriophage T4, the gamma complex in E. coli, and replication factor C (RF-C) in eukaryotic cells (O’DONNELL et al. 1993). In each case, the clamp loader is a multiprotein complex that has several characteristic activities in vitro. RFC, for example, is a multisubunit DNA-binding protein that interacts with the 3‘-OH terminus at primer-template junctions (LEE et al. 1988; YODER and BURGERS 1991; FIENand STILLMAN 1992; PODUSTet al. 1995). In addition to binding DNA, RF-C also associates with PCNA, and it can load PCNA onto the double helix in an ATP-dependent reaction (LEE and HURWITZ 1990; TSURIMOTO and STILLMAN 1991). Although biochemical evidence suggests that this reaction is important for processive DNA replication in vitro, the role that these proteins play in DNA replication and DNA repair in vivo remains to be determined. In yeast, the RF-C complex is composed of five different subunits (one large and four small),each of which is encoded by an essential gene (HOWELL et al. 1994; CULLMAN et al. 1995). Allfive of the subunits contain a conserved 225-amino acid domain (RF-C box) that includes a nucleotide-binding consensus sequence. The four small subunits range in sizefrom 323 to 354 amino acids and share ahigh degree of sequence similarity to one another. In contrast to the small subunits of RF-C, the large subunit is 861 amino acids long. In addition

66

M. A. McAlear, K M. Tuffo and C. Holm

to containing the conserved RF-C box, it also includes an additional 330-amino acid Gterminal domain and a 300-amino acid N-terminal domain, part of which has homology to sequences found in prokaryotic DNAligases. Thus, the large subunit is likely to play a distinct role in contributing toRF-C function. Although studies on both human and mouse RF-C indicate that the large subunit can bind to DNA on its own in vitro(TSURIMOTO and STILLMAN 1991; BURBELOet al. 1993), the precise role that this protein plays in DNA metabolism in vivo is not clear. In this study, we demonstrate that the large subunit ofRF-C (Cdc44p or Rfclp) is required for both DNA replication and DNA repair in vivo. When cdc44 mutants are released from an alphafactor arrest at the restrictive temperature, they progress through S phase very slowly, and they ultimately arrest with a G2/M DNA content before the initiation of mitosis. cdc44 mutants also exhibit a mutatorphenotype in vivo,and they are sensitive to MMS and W irradiation. In addition, we demonstrate that after irradiation with UV light, cdc44 mutants have a higher level of single-stranded DNA breaks than do CDC44 cells. Taken together, these results suggest that Cdc44p plays an essential role in therepair of single-stranded breaks in DNA. MATERIALSAND METHODS

Strains and media: The yeast strains used in this study are listed in Table 1. Standard genetic techniques were used in et al. 1986), and the conconstructing the strains (SHERMAN struction of all strains created by integration of plasmid DNA was verified by Southern analysis (SOUTHERN 1975). To create rho" isolates of strains, rho+ strains were first inoculated into liquid W D medium containing 25 yg/ml of ethidium bromide and incubated for 2 days at 30" (SHERMAN et ul. 1986). The cultures were then streaked onto W D plates and incubated at 30" for a further 3 days. Single colonies were recovered from the plates and tested for their ability to grow on YEP medium with glycerol (2%) as a carbon source. Rhocolonies were tested for the presence of mitochondrial DNA by staining the cells with 4',6-diamidino-2-phenylindoleand examining the cells by fluorescence microscopy. Strains CH2213 and CH2214 were constructed by transforming strain MKF"o with a DNA fragment containing either the WC44 orcdc44-I gene thatwas linked to a DNA fragment containing the HIS3 gene. Briefly, for the construction of CH2213, a 1.8-kb BamHI fragment of the HZS3 gene derived from plasmid pJJ215 (JONES and PRAKA~H 1990) was inserted into the SnaBI site downstream of the WC44 genein plasmid plasmid pCH1335 (HOWELLet al. 1994).Thiscreated pCH1541, which contained the HZS3 gene attached downstream of the CDC44 gene. A 4.8-kbStuI-KpnIWC44.HZS3 fragment from pCH1541 was then used to transform the his3 strain MKF"o, creating strain CH2213 (WC44.HIS3). An identical strategy using the cdc44-I plasmid pCH1350 was used to create strain CH2214 (cdc44-1.HZS3). Plasmids pCH1587 (PGALI-WC44) and pCH1588 (pGAL1cdc44-I) were created by cloning a Fnu4HI-SnuBI WC44 fragment from plasmids pCH1335 (WC44) andpCH1350 (cdc44I ) into the SmuI site of plasmid pCH1155 (PGALI) (KRANz 1993).A NotI fragmentfrom pCH1587 or pCH1588 was

cloned into the NotI site of plasmid pCH1099 (URA3) to create plasmids pCH1523 (PGALI-WC44 URA3) and pCH1524 (pGAL1-cdc44-IURA3).These plasmids were transformed into strain CH1587 (ura3-52 trpl WC44::LEU2 [pCH1289 ( W C 4 4 TRPl)]1, and the resulting strains were streaked onto SD plus Trp medium. Trpisolates that hadlost the plasmid pCH1289 (WC44 TRPl) were recovered,creatingstrains CH2088 { WC44::LEUZ uru3-52 trpl [pCH1523(pGALl-WC44 URA3)]] and CH2089 (CDC44::LEUZ ura3-52 trpl [pCH1524(pGAZ,lcdc44-1 UR43)I). YEPD medium is 1% yeast extract, 2% bacto-peptone and 2% glucose. YEP Raf/Gal medium is 1% yeast extract, 2% bacto-peptone, 2% galactose and 2% raffinose. SD medium is 6.7 g/I yeast nitrogen base without amino acids (Difco), 20 g/l of glucose, and 20 g/1 of bacto-agar (Difco). SC medium isSD medium supplemented with 20 mg each of adenine, histidine, and uracil, 60 mg of leucine, 30 mg of lysine, and 20 mg of tryptophan per liter of medium. Molecularbiology: All standard techniques of molecular biology were performed as described (AUSUBEL et al. 1988). Creation of new cdc44 alleles: New mutant alleles of the CDC44 gene were created by random or sitedirected mutagenesis. For random mutagenesis, the plasmids pCH1530 (WC44.LEU2 URA3) and pCH1531 (CDC44.LEU2 URA3) were propagated in the mutagenic mutD E. colistrain KD1067 (arg his mutD5 Sut) (HENKINand SONENSHEIN 1987). Mutagenized plasmids were recovered from E. coli and then transformed into the yeast strain CH1587. The plasmid shuffle technique (BUDD andCAMPBELL 1987) was used to select for strains bearing only the mutagenized plasmids, and these strains were screened for mutant phenotypes. Sitedirected mutations in the WC44 gene were constructed by the method of KUNKELet al. (1987). Mutant plasmids were recovered from E. coli and sequenced to confirm the presence of the mutations, and the mutant WC44fragments were subcloned into plasmid pCH1335. The new mutant WC44alleles were transformed intoyeast strains bearing a deletion in the WC44 gene by a plasmid shuffle method (BUDD and CAMPBELL 1987). Transformants were screened for phenotypes including heat and cold sensitivity, and sensitivity to MMS and hydroxyurea. GST-Cdc44p fusion proteins and GTP binding assays: To purify Cdc44p for GTP binding assays,we created GSTCdc44p fusion constructs and purified the fusion proteins with glutathione agarose beads. Briefly, a 3.4kb BamHI-EcoN WC44 fragment fromplasmid pCHl335 (CDC44) was cloned into plasmid pGEX-1X (SMITHand JOHNSON 1988) that was digested with BamHI and EcoN. This created plasmid pCH1484 (GST-WC44). A similar strategy using plasmid pCH1350 (cdc44-1) was used to create plasmid pCHI499 (GSTcdc44-I). In these constructs, the nonessential N-terminal 140 amino acids of the WC44 gene (HOWELLet ul. 1994) were replaced with sequences encoding the GST protein. Plasmids were transformed into E. coli and grown overnight, and expression of the fusion proteins was induced with 1 mM isopropyl thiogalactoside for 3 hr. Cells were collected, digested with lysozyme (0.2 mg/ml), and lysed by sonication, and the lysates were incubated with glutathione agarose beads (Sigma). The glutathione beads were washed with buffer A (phosphate-buffered saline with 20% glycerol, 1% NP-40, 1 mM EDTA, 1 mM 1,4dithiothreitol, 1 pg/ml leupeptin, 1 yg/ ml pepstatin and 1 mM phenylmethyl sulfate fluoride) to remove unbound proteins. The presence of the fusion proteins was confirmed by SDSPAGE. Equal amounts of protein were then incubatedwith alpha jnP GTP (NEN duPont) for 10 min at 30" in buffer B (50 mM Tris pH 7.5,50mM KCI, 5% glycerol and 0.5 mM MgCI2).After incubation, thebeads were washed

RFC in DNA Replication and Repair

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TABLE 1

S. cereuisiQe strains used in this study Strain CH325 CH526 CH533 CH541 CH545 CH756 CH1491 CH 1492 CHI 494 CH1587 CH1589 CH1591 CH1634 CH1806 CH1807 CH2027 CH2088 CH2089 CH2211 CH2212 CH22 13 CH2214 CH22 15 MKP-O

Genotype" MATa lys2-801 ura3-52 his4-539 top2-4 MATa ade 1 ade2 lys2 ural his7 tyrl gall cdc2-lb MATa ade 1 ade2 lys2 ural his7 tyrl gall cdc9-I MATa ade I ade2 lys2 ural gall cdcl7-1 MATa ade 1 ade2 lys2 ural his7 tyrl gall cdc21-lb MATa ade 1 ade2 lys2 ural his7 tyrl cdc8-1 MATa leu2-3,I 1 2 ura3-52 cdc44-1 MATa ade2 leu2-3,112 ura3-52 MATa ade2 leu2-3,112 ura3-52 cdc44-I MATa ura3-52 leu2AI trplA63 W44::LEUZ [pCH1289 ( W C 4 4 TRPI)] MATa ura3-52 bu2AI trplA63 W44::LEU2 [pCH1297 (cdc44-2 TRPI)] MATa ura3-52 leu2Al trplA63 W44::LEU2 [pCH1289 (cdc44-4 TRPI)] MATa his4 lys2 ura3-52 rad52::URA3 MATa ura3-52 his4-539 lys2-801 W C 4 4 .URAP MATa ura3-52 his4-539 lys2-801 cdc44-1.URAP MATa ura3-52 leu2 rad9::LEU2 MATa ura3-52 leu2Al trplA63 WC44::LEUZ [pCH1523 (pGAL1-WC44 URA3)] MATa ura3-52 leu2Al trplA63 WC44::LEU2 [pCH1523 (PGALI-cdc44-1 URA3)] MATa ade2 leu2-3,112 ura3-52 W C 4 4 [rho"] MATa ade2 leu2-3,112 ura3-52 cdc44-I [rho"] MATa canl-100 ade2-1 lys2-1 ura3-52 leu2-3,112 his3-A200 trpl-A901 WC44.HIS3 wCpMP2 SUP4-01 MATa canl-100 ade2-l lys2-1 ura3-52 leu2-3,112 his3-A200 trpl-A901 cdc44-l.HIS3 wCpMP2 SUP4-01 MATa ade2 ura3-52 cdc9rho" MATa canl-100 ade2-1 lys2-1 ura3-52 leu2-3,112 his3-A200 trpl-A901 IJ'CpMP21

HOLMet al. (1985) et al. (1973) et al. (1973) et al. (1973) HARTWELL et al. (1973) HARTWELL et al. (1973) M&.EAR et al. (1994) MCALEARet al. (1994) This study HOWELLet al. (1994) This study This study SPELLet al. (1994) HOWELLet al. (1994) HOWELLet al. (1994) This study This study This study This study This study This study HARTWELL HARTWELL HARTWELL

This study

KUNZet al. (1989)

Extrachromosomal elements and plasmids indicated by [ 1. This strain has additional unidentified auxotrophies. 'This strain has URA3 integrated next to W C 4 4 or cdc44-1 at the W C 4 4 locus. a

three times in buffer B to remove any unbound GTP and then counted in a scintillation counter. Syntheticlethality: Cold-sensitive strain CH1491 (cdc44-1) was crossed with each of the heat-sensitive strains CH526 ( c d ~ 2 - lCH756 )~ (cdca-l), CH533 (cdc9-l), CH541 (cdcl7-1), CH545 (cdc21-I) and CH325 (top2-4). Diploid strains were selected, grown on rich medium, and sporulated. Tetrads from eachcross were dissected, and the resulting spores were allowed to germinate on YEPD at 25". This temperature was permissive for both the cold-sensitive (Cs-) and heat-sensitive (Ts-) strains. The viability of the spores and the segregation of markers were determined for2 1 4 tetrads from eachcross. We were able to recover at least eight double mutant(Cs- Ts-) spores from eachof the diploids that were sporulated, except forthe diploidstrain that was derived fromthe CH1491 (cdc44) X CH526 (cdc2) cross. In that case, we recovered zero out of an expected 15 double mutant (cdc44 cdc2) spores. Detection of spontaneous SUP& mutants: Spontaneous mutagenesis assays were performed essentially as described previously (KUNZ et al. 1989). We used isogenic strains CH2213 ( W C 4 4 )and CH2214 (cdc44-1) that carried chromosomal canl-100, ade2-1, lys2-I and ura3-52 alleles. The canl100, ade2-1, lys2-1 alleles were suppressed by a SUP4-0 ochre suppressor tRNA gene that was present on an extrachromosomal YCp URA3, SUP4-o plasmid. Cells harboring the SUP4o plasmid were therefore Can', Ade+, Lysf, and Ura+. Cells acquiring a mutation in the SUP4-0 gene become Can', Ade-, Lys-, and Ura'. Strains CH2213 and CH2214 were streaked for single colonies onto SC "ura plates to maintain the URA3

at for 4 days. IndepensUP4-o plasmid and then incubated 30" dent colonies from each strain were used to inoculate 5 m l cultures of liquid SC "ura media, and these cultures were incubated overnight at 30". Cells were sonicated, counted, diluted as necessary in water, and plated onto SC "ura, SC "ura plus canavanine (30 mg/ml), and YPD plates. Plates were incubated at 30" for 5 days and then scored for colony growth. For each strain, the data from 15 independent cultures were used to determine the mean mutation frequencies. Red (ade-) canavanine-resistant colonies were tested for lysine auxotrophy, and Can', Ade-, Lys- clones were retained for sequencing analysis. For sequencing, PCRwas used to amplify a DNA fragment containing theSUP4-0 gene directly from yeastcells. The primers used for this amplification flanked the SW4-o gene and were of the following sequence: primer 1,5'-GTGGCGCCGGTGATGCCGGG3'; primer 2,5'GTTGTGTGGAATTGTGAGCG-3'.The amplified DNA product was used as a substrate for sequencing by the fmol'" DNA sequencing system (Promega). Microfluorometric analysis: Strains CH1806 ( W C 4 4 ) and CH1807 (cdc44-1) were grown to early log phase in liquid W D p H 4 at 30" and then arrested with alpha factor (5 pg/ ml Sigma) for 3 hr. Cells were then washed free of alpha factor and released into fresh YPD at the permissive (30") or restrictive (14") temperature. At the indicated times, cells were collected and fixed in ethanol. Cells were then treated with 1 mg/ml RNAseA, stained with 50 pg/ml propidium iodide, and processed for microfluorometric analysis on a Becton Dickinson FACScan fluorescence-activated cell sorter

68

M. A. McAlear, K. M. Tuffo and C. Holm

(HUTTER and EIPEL1979). Each DNA content histogram was based on the analysis of 210,000 cells. Reciprocal shift experiments: Strains CH1806 ( W C 4 4 ) and CH1807 (cdc44-1) were grown to early log phase in liquid YPD medium at30"and then treatedas indicated. For control experiments, a sample of the 30" culture was plated onto 14" YEPD plates or 3O"YEPD plates containing 0.3 M hydroxyurea. Although control cultures were also plated onto YEPD plates containing 80 pg/ml benomyl, only about half of the cells arrested as doublets, makingthe results of this shift impossible to interpret; results of shifts to benomyl plates are therefore not reported here. For the experimental cultures, cells were first incubated in YPD containing HU or nocodazole at 30" for 3 hr, or in YPD alone at 14" for 12 hr. The cultures were then chilled, washed, sonicated, and plated onto YEPD plates at 14" or onto 30"YEPD plates containing HU. Cell morphology was scored immediately by light microscopy (zero time) and then again after either 5 hr at 30" or 12 hr at 14". Sensitivity to DNA damaging agents: For assaying sensitivity to DNA damaging agents, yeast strains were first grown in liquid YPD medium at 30" to early log phase, sonicated, and diluted in water as necessary. For assaying UV sensitivity, the cells were plated onto YPD plates and exposed to up to a maximum of 200 J/m' of 254 nm UV radiation from a model 1800 Stratalinker (Stratagene). For assaying MMS sensitivity, cells were incubated in 0.3%MMS for up to 40 min and then plated onto YPD plates. For assaying sensitivityto gamma rays, cells were diluted in water, exposed to u to a maximum of 20 krads of gamma radiation from a Cs' source, and then plated onto YPD plates. Plates were incubated at 30" for 3 days and then scored for viability. The plots represent the average viabilities determined from two independent experiments. Velocitysedimentation gradients: Cell culture and chromosomal DNA isolation was performed by a modification of a previously published protocol (BUDDet al. 1989). Briefly, strains CH2211 [WC44(rhoo)]and CH2212 [cdc44(rhoo)] were grown in liquid SC at 30" to a density of -5 X lo6 cells/ ml.Sixty millicuries of5,G'H uracil (35Ci/mmol) (NEN Dupont) or 10 mCi of I4C uracil (60 mCi/mmol) (NEN Dupont) were added to 500 p1 aliquots of cells, and aliquots were incubated for 6-8 hr at 30".Where indicated, cultures were irradiated with 100 J/m' W radiation or shifted to 14". (At 100J/m' of UV irradiation, the viability ofthe cDC44 and cdc44 cells was 80 and30%,respectively.) After incubation, the cells were pelleted, resuspended in 0.5 ml of cold 0.1 M Tris pH 8.5, 0.1 M EDTA, 2% 2-mercaptoethanol, and incubated on ice for 15 min. Cells were washed, resuspended in cold 0.01 M KP04pH 9.72, 0.01 M EDTA and then transferred to the bottom of a 5-ml polyallomer centrifuge tube (Beckman). Twenty microliters of 10% zymolyase and 10 pl of RNAse A (10 mg/ml) were added to the cells, and the samples were incubated at 37" for 15 min. Nonidet P40 and sarkosyl were added to a final concentration of 2%, and for the alkaline gradients, NaOH was added to 0.5 M. A 4ml 15-30% sucrose gradient with 0.7 M NaCI, 0.03 M EDTA (and 0.3 M NaOH for alkaline gradients) was pumped into the bottom of the tube. Alkaline gradients were centrifuged for 17 hr at 12,000 rpm in a Beckman SW55 rotor at 20"; neutral gradients were centrifuged at 12,000 rpm for 13 hr.After centrifugation, the bottoms of the tubes were punctured, and20 200-pl fractions were collected. For the neutral gradients, residual RNAwas hydrolyzed by incubationovernight with the addition of NaOH to 0.5 M. Refractive indices of each fraction were determined to confirm the integrity of each gradient.Aliquots (150 pl) from each fraction were precipitated with 3 ml of 5% trichloroaceticacid, 0.1 M Na pyrophosphate.Precipitates

r'

were collected onto GF/C filters (Whatman), and the filters were washed with 25 ml 5% trichloroacetic acid, 0.1 M Na pyrophosphate, and 10 ml ethanol. Filters were counted for H and 14Cin 5 ml Ecoscint (National Diagnostics) for 5 min each. To eliminate the contributionsof mitochondrial DNA from this type of analysis, we used rho" isolates from each strain tested(SHERMANet al. 1986). To facilitate the comparison between the profiles of different strains, two chromosomal DNA preps (ie., one from W C 4 4 cells labeled with 'H uracil and one from cdc44 cells labeled with 14C uracil) were run together within the same gradient. The resulting profiles were confirmed by repeating the experiments with the strains labeled in the opposite orientation. The profiles are plotted with fraction 1 representing the top of the gradient and fraction 20 representing the bottom of the gradient. DNA from bacteriophage T4 was sedimented in parallel gradients and used as a molecular weight marker. Under neutral gradients, T4 DNA migrated to fraction 5. Under alkaline conditions, T4 DNA migrated to fraction 6. To calculate the number-average molecular weight (M,) of the gradient profiles, we used the formula (GREENet al. 1974; BUDDet al. 1989)

C, M, = -

x-MtC, t

where C, is the activity in the ith fraction. M, is computed by the equation (FRIEFELDER 1970) MZ/M*4 = ( d,/ d ~ )4''69, where d,is the distance of the ithfraction and h4is the distance sedimented by T4 DNA (MT4= 160 kb). Since the very top of the gradients do notresolve DNA fragments well, the contributionsof the first three fractions from each profile were omitted from thecalculation of the number-average mclecular weight (PETESand FANCMAN 1972). RESULTS

Genetics of cdc44 mutations: To characterize essential domains and activities of the large subunit of RF-C, we used a genetic approach to isolate and characterize W C 4 4 mutants. Analysis of the W C 4 4 sequence reveals that it contains a central225-amino acid putative nucleotide-binding domain that is conserved among allof the subunits of RF-C (RF-C box), and a N-terminal region that shares sequence homology with E. coli DNA ligase (Figure 1A) (HOWELL et al. 1994; CULLMANet al. 1995). Previously, we reported four conditional coldsensitive cdc44 alleles, each of which contained mutations within the RFC box. Using a differentmutagenesis procedure (mutD mutagenesis), we recovered four new cdc44 alleles (cdc44-I 0- cdc44-13), all of whichare sensitive to the alkylating agent MMS, and threeof which are cold-sensitive (Figure 1B). Three of the new mutants containmutations within the RF-C box, suggesting again that this is a domain within the Cdc44 protein that is important for its function. The fourth mutation lies within the DNA ligase homologous domain, indicating that this region, too, is important for Cdc44p activity.

RFC Repair in DNA and Replication

A

I 0

IligaseI

I

I

RF-C Box

I

I

I

I

I

I

I

I

100

200

300

400

500

600

700

800 861

B Amino acid

chanee

Allele

cdc44-1 cdc44-2' cdc44-4' cdc44-9 cdc44-10 cdc44-11 cdc44-12 cdc44-13 cdc44-14 cdc44-15 cdc44-16

D513N G428H

G436R G512A,D513N G185E. P234L Fs. V437 G356S Fs. K619 P355A T510H T386G

Phenotwe

Location

Cs-, MmsCs-, MmsCs-, MmsCs-, MmsCs-, MmsCs-, MmsCs', MmsCs-, MmsCs', Mms* Cs*, Mms' Cs', Mms'

P,C P P P This P P P P P P P

Ref.

Howellet

al. (1994)

study

FIGURE1.-Mutant W C 4 4 alleles. (A) Mapof the CDC44 gene indicating the regionsof homology to the smaller subunits of replication factor C (RF-C box) and to E. coli DNA ligase (ligase). (B) Listof cdc44 alleles and their properties. Fs., frameshift mutation; Cs-, cold sensitive; Mms-, methylmethane sulfonate sensitive;P, phenotype detected ina strain carrying a plasmid-borne mutation;C, phenotype detectedin a strain carrying a chromosomal allele; I , mutations in truncated alleles.

We also tested the significance of a putative nucleotide-binding domain that contains sequence motifs that are similar to GTP-binding consensus sequences. It had appearedthat these motifs might be significant, because three previously isolated cdc44 alleles contained mutations in or near theputative consensus sequences. In particular, a p2lRAS mutation analogous to the cold sensitive cdc44-1 mutation decreases p21- binding of guanine nucleotidesby 100-fold (CHANNINGet al. 1986). First, we tested Cdc44p for GTP binding directly. We expressed GST-tagged Cdc44p in E. coli, purified the recombinant protein, and assayed it for GTP binding in vitro. Although we could detect GTP binding in a GST-p21RAS control protein, we could not detect GTP binding in eitherGST-Cdc44por GST-Cdc44lpfusions (data notshown). Second,we constructed site-directed mutations within the putative GTP-binding domain and tested the resulting alleles for mutant phenotypes. We created three new cdc44 alleles, each of whichwas based on a known mutation that disrupts the activity of the GTP-binding protein ~ 2 1These ~ . alleles included mutations in the putative G1 region (P355A), G2 region (T386G) and G4 region (T510H) of Cdc44p. The analogous mutations inp2lW affect GTP hydrolysis,~ 2 activation, and nucleotide binding,respectively (VALENCIA et al. 1991). Although we looked for sensitivities to heat, cold,hydroxyurea, MMS and UV irradiation, none of these alleles gave rise to any detectable mutant phenotype. Taken together, these results suggest that it is unlikely that the essential activity of Cdc44p requires GTP-binding. Finally, we used synthetic lethality to identify proteins

69

involved in DNA metabolism with which Cdc44p might interact in vivo. We crossed a cdc44 mutant strain with several mutants defective in DNA metabolism. We foundthat cdc44-1 exhibited synthetic lethality with cdc2-1 (DNA polymerase 6) but not with cdc8-1 (thymidylate kinase), cdc9-l (DNA ligase), cdcl7-1 (DNA polymerase a), cdc21-1 (thymidylate synthase) or t@2-4 (DNA topoisomerase 11). Although other interpretations are possible, these results are consistent with the hypothesis that Cdc44p interacts directly with DNA polymerase 6 in vivo, and they are consistent with the biochemical evidence that RF-C interacts with DNA polymerase 6 in vitro (TSURIMOTO and STILLMAN 1989; BURGERS1991). cdc44 mutants exhibita nonspecific elevationin mutationfrequency: Although cdc44 mutants have an elevated mutation rate (MCALEARet al. 1994), it was not clear whether this mutator phenotype isof a general nature, or if cdc44 mutants have increased levels of a specific typeof DNA mutation. To address this question, we took advantage of an assay that has been developed to identify and quantitate spontaneous mutations that arise within a small plasmid-borne reporter gene (KUNZ et al. 1989). Isogenic strains CH2213 (CDC44) and CH2214 (cdc44-1) were constructed bearing the chromosomal canl-100, ade2-1, lys2-1 and ura3-52 alleles, as well as a nonessential, extrachromosomal, centromere plasmid-bearing URA3 and SUP4-o. In these strains, SUP4-o supresses the canl, ade2, and lys2alleles,resulting in cells that are phenotypically Can', Ade+, and Lys+. Mutations arising within the 89-bp s i P 4 - o gene produce cells that are Can', Ade-, and Lys-, and they can be selected by plating the cultures on plates containing canavanine (KUNZ et al. 1989). We observed thatfor the CDC44 strain, the mean frequency of canavanineresistant mutants was 0.8/106 cells (Table 2). This frequency is similar to the value (2.0/106 cells) that was previously reported as the mean mutation frequency for the SW4-o gene in the parental wild-type strain MKP-o (KUNZet al. 1989). In contrast, the cdc44-1 strain exhibited a mean mutation frequency of 12/106 cells. This 15-fold increase is consistent with our previous analysis of mutation frequencies in the 1.8-kb CAN1 gene in CDC44 and cdc44 strains (MCALEARet al. 1994). To examine the sequence-specificity of the mutator phenotype associated with cdc44 mutations, we sequenced 30 independent s i P 4 - o mutations that arose 1from~either CDC44 or cdc44-I strains. As had been observedpreviouslywith the wild-type parent (MKP-o) (KUNZ et al. 1989), the majority of the mutations in both the CDC44 and cdc44-1 cultures were single bp substitutions (87 and 83%, respectively) (Table 2). When the mutations were divided into the classes of transitions and transversions, a two by two contingency table analysis revealed that the spectrum of the mutations did not vary significantly between the CDC44 and

M. A. McAlear, K. M. Tuffo and C. Holm

70

TABLE 2 Spectrum of nucleotide changes in cd& WC44

Frequency of Canr/106 cells Total no. of mutants examined % of mutants with single basepair substitutions Breakdown of basepair substitutions Transitions (%\ G C A-?: 20 A-T G C Total Transversions (%) G C T-A G C C-G A-T + C-G 3 A-T T-A Total

0.8 30

-+

-+

-+

-+

a

cdc44-1

MKP-0"

12 222 30

2.0

87

83

82

29 14 43

17 37

17 -

21 29 4 4 58

-+

and CDC44 strains

25 42

43

32 19 2 6 59

7 10

63

"

As reported by KUNZ et al. (1989).

cdc44-1 strains ( P < 0.05). We did, however, observe that the cdc44-1 strain exhibited a doubling in the frequency of G C to T-A mutations, suggesting that the mutator phenotype might be related, in part, to the metabolism of G C base pairs. In both W C 4 4 and cdc44 strains, the positions of the mutations were distributed throughout the SUP4-o gene and were reminiscent of the positions previously reported for thewild-type MKPo strain (Figure 2) (KUNZet al. 1989). Thus, although cdc44-1 mutants exhibitan increased frequency ofspontaneous mutations,these results show that the spectrum of the mutations is not dramatically different thanthose observed in W C 4 4 strains. cdc44 mutants are delayed in progressing through S phase: Since Cdc44p is an integral component of the DNA replication apparatus, an increased mutation frequency could arise from defects in DNA replication or DNA repair. Although RF-C has been shown to be required for processive DNA replication invitro, the role it playsin DNA synthesis in vivo has not been determined. To determine whether RF-C isrequired forDNA replication invivo, we monitored the progression of DNA synthesis in synchronized CDC44 (strain CH1806) CUI244

6 hours 8 hours

1 2 hours

FIGURE 3.-"icrofluorometric analysis of CDC44 and cdc44 strains. Strains CH1806 (CDC44) and CH1807 (cdc44) were grown to early log phase at 30", arrested in G1with alpha factor, and then released from arrest at either 30" or 14". Samples were fixed, stained with propidiumiodide and counted by fluorescence activated cell sorter.

and cdc44-1 (strain CH1807) cultures (Figure 3). Mutant and wild-type cells were arrested in G1 with alpha factor, released into fresh medium at the permissive or restrictive temperature, andmonitored forDNA synthesis by microfluorometric analysis. When CDC44 cells are released from alpha factor arrest at 30°, they proceed into S phase, and by 30 min one can detect cells with a G2/M DNA content (Figure 3A). When cdc44 cells are released at 30",they progress through the cell cycle with similar kinetics (Figure 3B). However, when these strains are released from alpha factor arrest into meA

T

A

C

A A

TCC A

GA

C

T T

T T

A

T A A

G

TA

T AG

AAC

3 ' GAGAGCCATC GGTTCAACCA ATTCCGCGTTCTGAAATTA AATAGTZA'E CTTTAGAACT CTAGCCCGCA AGCTGAGCGG GGGCCCTCT 5 ' 89 1 10 20 30 40 50 60 70 80 C G T T C AA C TC C C A T G C T G TC T T C A T C A T

CDC4 4

FIGURE2.-Distribution of spontaneous s m 4 - o mutations. The 89-base transcribed strand of the s m 4 - o gene is shown. The spontaneous mutations identified from the cdc44-1 strain are shown above the sequence, and those from the W C 4 4 strain are indicated below. Two independent deletion mutations from the W C 4 4 strain and three independent deletion mutations from the cdc44 strain are not shown. In each case, a single guanine was deleted from the run of five guanines from bases 79 to 83.

RFC in DNA Replication and Repair

diumat 14", the cdc44 mutants progress through S phase very slowly. Whereas the majority of the CDC44 cells have a G2/M DNA content 2.5 hr after release from alpha factor (Figure 3C), cdc44 mutants only begin to accumulatecells witha G2/M DNA content 8 hr after release from alpha factor (Figure 3D). Theincrease in DNA content in cdc44 cells is not due to anaccumulation of mitochondrial DNA, because similar results were obtained with strains CH2211 (CDC44) and CH2212 (cdc44), whichlack mitochondrial DNA (datanot shown). Theseresults demonstrate thatcdc44-I mutants experience a delay in traversing S phase at the restrictive temperature. To rule out thepossibility that cdc44 mutants areable to traverse S phase because of an aberrant function of Cdc44lp at the restrictive temperature, we depleted Cdc44p from cells using transcriptional inactivation in cells grown only at the permissive temperature. We engineered strains that could be depleted of Cdc44p by regulating its expression with the repressible GAL1 promoter (PGAL).The plasmids pCH1523 (PGALI-CDC44) or pCH1524 (pGAL1-cdc44-I) were transformed into a CDC44 deletionstrain, andthe transformants were maintained on galactose (inducing)media.Unfortunately, thedepletionexperimentcould not be performed with the strain (CH2088) bearing the PGALCDC44 construct, because the residual transcription of the pGAL-CDC44 construct allowed the cells to grow even in the presence of glucose. However, the CDC44 deletion strain carrying the PGAL-cdc44-1 plasmid (CH2089) grew on galactose medium but not on glucose medium,indicatingthat Cdc44p could be depletedfromthe cells by growing them on glucose. PGAL-cdc44-1 cells were grownto early log phase under inducing conditions (in W raffinose containing galactose) and then shifted to medium containing glucose to repress the expression of the cdc44-1 gene. Consistent with the earlier temperature-shift results, PGAL-cdc44-1 cells arrested with a G2/M DNA content beforemitosis (datanotshown).Thus it appearsthatalthough Cdc44p is normally involved in DNA replication, it is nonetheless possible for the cell to complete the bulk of DNA replication even after Cdc44p activity drops below a critical level. Cdc44p activity is required to traverse the G2/M boundary: Regardless of the method used for reducing Cdc44p activity in the cells, cells with reduced Cdc44p activity always undergo a cell cycle arrest with a G2/M DNA content (Figure 3 ) (HOWELLet al. 1994). This result demonstratesthatalthough normal levels of Cdc44p activity are not essential for the bulk of DNA synthesis, theyare required forsome process that allows the cells to continue with the cell cycle. To determine whether Cdc44p has an activity that is required at the end of S phase, we examined the time of action of the Cdc44p function that is inactivated by the cdc44-I muta-

71

tion. We used reciprocal shift experiments to determine the order of the Cdc44p execution point with respect to other defined points within the cell cycle. In these experiments, cdc44 cells are alternately arrested and released from arrest under various conditions, and the progression of the cells through the cell cycle is determined by monitoring cell morphology UARVIK and BOTSTEIN1973; HEREFORD and HARTWELL 1974). First, we investigated the relationship between the execution points of Cdc44p and the drughydroxyurea (Table 3). Hydroxyurea is a reversible inhibitor of the enzyme ribonucleotidereductase, andtreatment of cells with hydroxyurea leads to a depletion of nucleotide pools, causing cells to arrest in S phase (SLATER 1973). When cdc44-I mutants are first arrested in hydroxyurea at 30" and then washed free of hydroxyurea and plated on W D plates at 14", they fail to progress through the cell cycle,remaining arrested as large budded cells. In contrast, if cdc44-I cells are first arrested at 14" and then placed onto hydroxyurea plates at 30", the cells progress through the first cell cycle and then arrest in the second cell cycle. Formally, these results demonstrate that the Cdc44prequiring step is dependent upon the HU-sensitive step. Thus, it appears that when cdc44 cells are arrested at 14", they are at a point in the cellcycle after the hydroxyurea-sensitive step, which is usually thought of as the bulk of DNA replication (SLATER 1973). This interpretation is consistent with the results of the microfluorometric analysisof synchronized cells. Second, we investigated the relationship between the execution points of Cdc44p and the drug nocodazole (Table 3). Nocodazole causes reversible depolymerization of microtubules, and cells treated with nocodazole arrest after the completion of S phase and at the beginning of mitosis (JACOBS et al. 1988). Unexpectedly, when cdc44-1 cells are first arrested at30" in nocodazole and then shifted into nocodazole-free medium at 14", the cells failto progress through the cell cycle. Because this result is unchanged if the initial growth and nocodazole arrest are performed at 35", [a temperature at which cdc44-1 mutants exhibit a normal FACS profile (data not shown)],it is unlikely that the choice of permissive temperature has any large influence on the experiment. This result suggests that nocodazole-arrested cells still havea requirement forCdc44p activity before they can continue through thecell cycle. However, only the cdc44-1 allele revealed this unusual execution point for Cdc44p activity;cells bearing the cdc44-2,cdc44-4, or cdc44-10 allele undergo a complete cell cycle after release from nocodazole arrest at 14" (data not shown), Although we cannot rule out the possibility that this result simply reflects a quirk of the cdc44-1 allele, it suggests that Cdc44p may play a role in the cell cycle beyond its role in DNA replication. Since the chromosomal DNA must be intact for cells to enter mitosis, one possibility is that Cdc44p plays a role in DNA repair.

M. A. McAlear, K. M. Tuffo and C. Holm

72

TABLE 3 Relationship between cdc44-1, hydroxyurea, and nocodazole execution points

incubation After

0 time

Treatment" Liquid plates Unbudded

Small bud Large bud

-+

1 or 2 cells

on plates

3 or 4 cells

>4 cells

0

0

86

14

Cell morphology (%)* wc44

30"

14" HU HU 14" 14" HU Noc -+ 14"

76 76 22 80 8

24 24 6 20 14

-+

30°C

+

-+

-+

0 0

77

0

0

0

80

16

78

0

0

100 0 100 4 100

12 42 8 74 20

0 0 0 4 0

cdc44-1

88

30" "* 14" 30°C HU HU 14" 14" HU Noc 14"

12

-+

92

-+

-+

+

64

12 12 6

58

0

20

80

76 76 30 20 10

12 80 70

22

a Strains CH1806 ( W C 4 4 ) and Ch1807 (cdc44-1) were grown exponentially in liquid mediumat 30°, treated for 1.5 generation times as indicated under liquid, sonicated, and then plated onto W D plates or W D plates containing HU. The morphologies of the cellswere determined immediately before plating (0 time) and again after 5 hr at 30" or 16 hr at 14". Unless indicated otherwise, the cells were incubated at a temperature of 30" and in the absence of any drug. Each regimen was repeated at least twice; representative results are

shown. "The number of cell bodies in each microcolony was determined by phasecontrast microscopy of cells on plates.

cdc44 mutants are defective in repairing singlestranded DNA breaks: If Cdc44p plays a role in DNA repair in addition to its role in DNA replication, then cdc44 mutants might be sensitive to one or more genotoxic agents. To test this hypothesis,we assayed whether cold-sensitive cdc44 mutations were associated with an increased sensitivityto the DNA-damaging agent MMS or to exposure to UV or gamma radiation. These genotoxic agents were chosen for their ability to induce a range of DNA damage, including chemically damaged DNA bases (MMS and W), and double-stranded DNA breaks (gamma radiation). Each of the cdc44 mutants tested had a 10- to 100-fold greater sensitivity to MMS exposure thandidthe CDC44 strain (Figure 4A). Sensitivityto UV irradiation was milder: the cdc44-1 allele caused the greatest sensitivity, -10 times greater than W C 4 4 ; cdc442 and cdc44-4 exhibited no significant increase in UV sensitivity (Figure 4B). None of the cdc44 strains were particularly sensitive to gamma irradiation (Figure 4C). Taken together, these results suggest that Cdc44p may play a role in the repair of damaged DNA bases but not in the repair of double-stranded DNA breaks. During both DNA replication and the repair of damaged DNA bases, single-stranded gaps are produced in the DNA, and these gaps must be processed to yield DNA. cdc44 mutantsmight intactdouble-stranded therefore be defective in the processing of singlestranded regions ofDNA. To test this hypothesis, we assayed for single- and double-stranded breaks in chromosomal DNA by using velocity sedimentation analysis

with neutral and alkaline sucrose gradients. We used strains lacking mitochondrial DNA for this analysis to ensure that theeffects we observed were due entirely to shifts in the state of chromosomal DNA. Strain CH2211 (CDC44) was labeled with 3H uracil, and strain CH2212 (cdc44-I) was labeled with 14C uracil. To facilitate the comparison between the profiles of W C 4 4 and cdc44 strains, DNA from both strains was sedimented within the same gradient. Chromosomal DNAwas isolated from both strains, and thesamples were sedimented on 15-30% neutral or alkaline sucrose gradients. Doublestranded breaks in the DNA would be revealed by altered sedimentation on both neutral and alkaline sucrose gradients. Single-stranded breaks would be revealed by altered sedimentation on alkaline gradients. We first compared chromosomal DNA from exponentially growing CDC44 and cdc44 strains at permissive or restrictive temperature. When the strains werelabeled at the permissive temperature of 30°, the sedimentation profiles of the DNA from thetwo strains were indistinguishable in both neutral and alkaline gradients (Figure 5A). For the alkaline gradient profiles, the number-average molecular weight was calculated to be 3.1 X 10'. For the neutral gradient profiles, the numberaverage molecular weight was 6.9 X 10'. This value for the neutral gradients is consistent with the known sizes of yeast chromosomes and is similar to the value (6.2 X 10') obtained by PETESand FANCMAN for yeast chro1972). To examine mosomal DNA (PETESand FANCMAN the DNA from cells grown at restrictive temperature,

73

RFC Repair in DNA and Replication

A

A-

-

loo0

-

5

ae

.1: 0

,

. ,

10

20

.

.

.

, 30

,

C

W

cdc44-1 cdc44-2

cdc44-4 rad9

I

40

Minutesin 0.3% MMS

B

I -

-

loo0

.1

1,

,

'

0

.

,

40

.

,

.

120

80

UV irradiation

, 160

~

I,

~

,

C

W

Cdc44-1

cdw-2 cdc44-4 rad9

~

200

(Jim')

C lorn

I

-

._ n a 5

in the alkaline gradient profile toward the top of the gradient (equivalent to a decrease in the number-average molecular weight by 30%, which corresponds to a 43% increase in the levelof single-stranded DNA breaks). The neutral sedimentation profile of the cdc9 mutant at therestrictive temperature was similar to the profile at the permissive temperature. Thus, although we could easily visualizesingle-stranded DNA breaks in a cdc9 mutant, much less breakage must be present in cdc44 mutants. Although any difference between the levels of DNA breaks in unperturbed c1DC44 and cdc44 cells must be low, we reasoned that it might be possible to visualize increased levels of DNA breaks in cdc44 cells following exposure to a genotoxic agent if Cdc44p plays an essential role in DNA repair. To examine this possibility, we determined whether cdc44 mutants had elevated levels of DNA breaks after exposure toUV light. When strains CH2211 (CDC44) and CH2212 (cdc44) were treated with 100 J/m' of UV irradiation, allowed to recover at 23" for 5 min, and then collected for sedimentation analysis, the alkaline DNA sedimentation profiles of both strains were shifted toward the top of the gradient (Figure 6A). In separate gradients in which irradiated and unirradiated samples from a given strain were run together (data not shown), we found that the number average molecular weight of the DNA decreased by only 10% for O C 4 4 cells. In contrast, the number-average molecular weight of the DNA decreased by 30% in the cdc44 samples. This result may reflect the rapidity with which CDC44 cells can repair W damage, and it suggests that cdc44 mutants may be deficient in W repair. To determine whether cdc44 mutants aredeficient in W repair at the restrictive temperature, we examined the kinetics of recovery at both permissive and restrictive temperatures. DNA breaks induced by UV irradiation were repaired if the cultures were irradiated and then allowed to recover at permissive temperature (30") before collecting for sedimentation analysis.For the CDC44 strain, the alkaline gradient profile showed that the DNA had returned to unirradiated levels of breakage after 30 min of recovery at 30" (Figure 6B). Althoughthe recoveryof the cdc44 strain was slightly slower, both the cdc44 and W C 4 4 strains exhibited normal profiles after 1 hr of recovery at 30" (Figure 6C) (data not shown). This result indicates that cdc44 mutants can repair damaged DNA when incubated at the permissive temperature. In contrast, when the strains were irradiated and then allowed to recover at 14" before sedimentationanalysis, recovery ofthe cdc44 strain was much slower; in fact, even after 21 hr the DNA had not fully recovered (Figure 6, D and E). Whereas the number-average molecular weight of the c1DC44 profile was only 5% lower than the unirradiated sample after incubation at 14" for 6 hr, the value for thecdc44 profile was 20% lower than the unirradiated sample (data not

10

.. , 0

-

;

~

I

10

15

~

CDW cdc44-1 cdc44-2 rad52

,

20

Gammairradiation(Krad)

FIGURE 4.-Sensitivity of strains to DNAdamagingagents. Strains CH1806 (CDC44, CH1807 (cdc44-I), CH1589 (cdc44-2), CH1591 (cdc44-4), CH1634 (rad52),and CH2027 (rad3 were grown to early log phase and exposed to the DNAdamaging agent as indicated. The cells were diluted as necessary, plated onto WD plates, incubated at 30"for 3 days and scored for growth of colonies. (A) Sensitivity to MMS exposure. (B) Sensitivity to UV radiation. (C) Sensitivity to gamma ray radiation.

we cultured D C 4 4 and cdc44 strains at the permissive temperature, shifted them to the restrictive temperature (14") for 10 hr, and then processed the samples (Figure 5B). Onceagain, we found that theCDC44 and cdc44 profiles were indistinguishable. Because we detected no difference between cdc44 and CDC44 strains, we examined a cdc9 (DNA ligase) mutant as a control for our sedimentation analysis. As expected, the sedimentation profile of strain CH2115 (cdc9) grown at the restrictive temperature (36") for 3 hr revealed a shift

~

,

74

M. A. McAlear, K. M. and Tuffo

C. Holm

Gradients Neutral Gradients Alkaline

A

0 5

0

10

15

20

0

5

Fraction

10

15

20

Fraction

B 4000

800

3000

600

5 n

I

;

P

2000

400 0

0

P r

1000

200

0

10

5

15

20

0

5

Fraction

500

300 -

100

400

-

300

-

200

-

60

-

100

-

40

20 ~

o ! 0

.

.

.

.

,

5

.

.

.

.

,

.

.

10 Fraction

.

.

I

.

.

.

15

.

!0 20

100

20

, 120 100

80

60 0

40

-

0 . 0

+

+

,

400

80

0

200

15

Fraction

C

2

10

R P

+

0 0

FIGURE 5 .-Velocitysedimentation analysis of chromosomal DNA. Strains were grown exponentially andlabeled with 'H or I4C uracil. Cells were then collected and lysed, and the DNA was sedimented on alkaline (left) and neutral (right) 1530% sucrose gradients. Gradient fractions were collected, reci itated and counted for'H and C. (A) Pro(CDC44, 0) filesofstrainsCH2211 and CH2212 (cdc44, ) growing at 30". (B) ProfilesofstrainsCH2211 (CDC44, 0) and CH2212 (cdc44, ) after incubation at 14" for 10 hr. (C) Profilesof strain CH2215 (cdc9) growing at permissive(23", 0) or restrictive (36", ) temperature for 3 hr.

2

20

. . . . . . . . . . . . . . . . . . .: 0 5

shown); the cdc44 mutant maintained a 20% reduction in number average molecular weight even after 21 hr at 14". This failure of cdc44 mutants to process UVinduced single-stranded DNA breaks at the restrictive temperature is consistent with the hypothesis that Cdc44p plays a role in theDNA excision repair pathway. DISCUSSION We report that the large subunit of RF-C (Cdc44p or Rfclp) is involved in both DNA replication and DNA repair in S. cereuisiae. We have characterized cold-sensitive mutant alleles of the CDC44 gene and demonstrate that when cdc44 mutants are released from an alpha factor arrest to the restrictive temperature, the mutant cells are delayed in progressing through S phase. cdc44 mutants also exhibit a general mutator phenotype, and they are sensitive to both UV irradiation and exposure

10

15

20 Fraction

to the methylating agent MMS. When strains are exposed to U V irradiation and then allowed to recover at 14", cdc44 mutant cells retain higher levels of singlestranded DNA breaks than do wild-typecells. These results, coupled with the known biochemical activities ofRF-C, are consistent with the hypothesis that RF-C plays a role DNA synthesis and is involved in themetabolism of single-stranded DNA gaps in both DNA replication and DNA repair in vivo. The phenotypes of cdc44 mutants suggest that Cdc44p activity is required both for efficient DNA synthesis and for the initiation of mitosis. When synchronized cdc44 cultures are released from a G1 arrest to the restrictive temperature, the synthesis of DNA proceeds very slowly, and the cells ultimately arrest in the cell cycle with a G2/M DNA content. This result indicates that Cdc44p is required for the normal progression of S phase. Previously, we demonstrated that when

75

RFC in DNA Replication and Repair

A 80

60

B0 40

CDC44 cdc44

-

0

P

20

0 155

0

10

20

Fraction

B

C 250

sedimentation FIGURE 6.-Velocity analysis of chromosomal DNA after UV irradiation. Strains CH2211 40 (CDC44, 0) and CH2212 (cdc44, ) grown were at 30°, irradiated with 100 3o 0 J/mz of UV irradiation, and then alp tolowed recover for 5 min at 23" (A), 30 min at 30" (B), 1 hr at 30" (C), 6 2o hr at 14" (D), or at21 hr 14" (E). Sampleswerecollected and lysed, 10 and the DNA was sedimentedon al20 kaline 15-30% sucrose gradients. 50

200

100 50 0

0

5

10

15

0

20

5

.""..

10 Fraction

Fraction

D

+

E

150

15

E ,

1100 120

600

-

6 hrs.

80

100 80

60

0

60

40

40

0

. . . . , . . . . , . . . . , . . . .! 0 51 5

10

20

0

20

20

o !

-

0 0

1 55

Fraction

an exponentially growing culture of cdc44 cells is shifted from the permissive temperature to the restrictive temperature, the cells also arrest with a G2/M DNA content, and that this cell cycle arrest is mediated by the m y d e p e n d e n t checkpoint (HOWELLet al. 1994). Thus, although the bulk of DNA replication can eventually proceed in cdc44 mutants at therestrictive temperature, it appears that unreplicated or damaged regions of the genome persist and prevent the cell cycle from progressing into mitosis. Cdc44p might therefore play a critical role in a final stage of DNA metabolism that ensures that the genome is in a state suitable for the initiation of mitosis. The sensitivity of cdc44 mutants to both MMS exposure and UV irradiation suggests that RF-C is involved inthe DNA excision repair process. The alkylating agent MMS produces 7-methylguanine (7MeG) and 3-

10

20

Fraction

methyladenine (3MeA), which are substrates forthe the base excision repair pathway (XMO and SAMSON 1993). UV irradiation leads to the formationof cyclobutane pyrimidine dimers and 6-4 photoproducts, each of which can be repaired by the nucleotideexcision repair pathway (reviewed in BOHR1991). The base and nucleotide excision repair pathways share similar mechanisms for repairing damaged DNA. In each case, the altered DNA bases are targeted for removal from the double helix. After a stretch of nucleotides including and surrounding thedamaged base is excised from the DNA, the resulting single-stranded gap is filled in by using the intact DNA strand as a template. Recently, it has been reported that both DNA polymerase 6 and PCNA are involved in the excision DNA repair process (BLANKet al. 1994; MATSUMOTO et al. 1994; BUDDand CAMPBELL 1995). Thus, it seems most economical to

76

M. A. McAlear, K. M. Tuffo and C. Holm

propose that after the excision of the damaged DNA strand, RF-C binds to the single-stranded gap and through its association with PCNA stimulates DNA polymerase activity. That RF-C functions in DNA repair through its interaction with PCNA is supported by the observation that PCNA (poZ30) mutations can suppress the DNA repair defects of cdc44 mutations (M. A. McALEAR and C. HOLM,unpublished data). What is not clear, however, iswhy the processivity factor PCNA is used in the polymerization ofvery short stretches of DNA. Perhaps in this context, the loading of PCNA onto the double helix is required for recruiting DNA polymerase to the site of DNA repair, rather than for increasing itsprocessivity (SHWJIet al. 1992; LI et aZ. 1994; ~ O U S S E K H R Aet al. 1995). That cdc44 mutants are not particularly sensitive to gamma ray radiation suggests that RF-C is not involved in the repair of double-stranded DNA breaks. While we have demonstrated that Cdc44p plays an important role in the repairof UV damage, theessential role of the protein in unirradiated cells is less clear. Velocity sedimentation analysis indicates that cdc44 mutants are compromised in their ability to process singlestranded DNA breaks atthe restrictive temperature. When W C 4 4 and cdc44 cells are irradiated with UV light and then allowed to recover at 14"C, the cdc44 mutants exhibit a higher level of single-stranded DNA breaks than do W C 4 4 cells. Thus, Cdc44p probably plays a role in the DNA excision repair process after the damaged bases are excised from the DNA duplex. It is puzzling, however, that unirradiated cdc44 mutants arrest before mitosis at 14". Since DNA replication proceeds slowly at 14" in cdc44 mutants, it would not be surprising if the integrity of the DNA were somehow compromised. Nevertheless, we were unable to detect a significant difference in the levels of DNA breaks in unirradiated CDC44 and cdc44 strains at 14". One possibility is that low levels ofDNA breakage may be present in cdc44 mutants, and that this damage precludes cells from enteringinto mitosis. It has recently been reported that spontaneous DNA damage can occur in highly transcribed regions of DNA (DATTAand JINKSROBINSON1995).Furthermore, it has beendemonstrated that as little as one double-stranded DNA break pergenome is sufficient to stop the cellcycle in S. cmeuisiae (BENNETTet al. 1993). Perhaps at the restrictive temperature of 14", the activity of the mutant Cdc44 l p is insufficient to deal with the final DNA gap-filling requirements created by the processes of DNA replication and DNA repair. Since RF-C is involved in both DNA replication and DNA repair, what is the source of the mutator phenotype associated with cdc44? Our results suggest that defects in both DNA repair and DNA replication could contribute to this phenotype. The spectrum of spontaneousmutations observed in cdc44 mutants suggests

that the mutations do not arise from a gross defect in DNA polymerization (i.e., DNA polymerase slippage, DNA insertions or deletions). Rather, the majority of the spontaneous mutations produced incdc44 mutants are single base pair substitutions. Although cdc44 mutants have a 15-fold increase in thefrequency of spontaneous mutations over CDC44 cells, the spectra of mutations found in the two strains are generally similar to oneanother.One potentially informative exception, however, is the increase in G C to T-A transversions in cdc44 mutants. When alkylated bases are removed from the DNA duplex and the subsequentabasic sitesescape DNA repair, there is a preferential incorporation of adenine opposite the abasic site during DNA replication (LOEB and PRESTON 1986). Thus,G C to T-A transversions could arise from the incomplete repairof damaged G residues (ROMOTAR et al. 1991). Spontaneous alkylation is a known source of7MeG in S. cereuisiae, and mutations in the enzymes that process 7MeG increase the frequency of spontaneous mutationby threeto fivefold (XIAOand SAMSON 1993). These results, coupled with the observation that the major form of damage induced by MMS is 7MeG, support the hypothesis thatthe cdc44-associated mutatorphenotype may be due in part to a defect in the base excision repair pathway. However, since mutants completely defective in 7MeG processing only have a three-to fivefold increase in mutation frequency (XIAOand SAMSON 1993), there must be an additional cause for the elevated mutation rate in cdc44 strains. Another source for the increased rate of mutation seen in cdc44 mutants might be errors produced during DNA synthesis due to disruptions in the usage of the DNA polymerases. In vitro biochemical analysis indicates that RF-C and PCNA stimulate the activities of DNA polymerases S and E (LEE and HURWITZ 1990; BURGERS1991; LEEet al. 1991;FIENand STILLMAN 1992; PODUSTet al. 1992). Of the five DNA polymerases in S. cereuisiae, these are the only two that have an intrinsic 3'-5' exonuclease proofreading activity (MORRISON and SUCINO1994). Therefore,if the DNA polymerase stimulating activities of RF-C and PCNA were compromised in cdc44 mutants, those processes that would normally be carried out by DNA polymerase S or E might be accomplished by another, potentially more error-prone polymerase. This disruption in DNA polymerase usage could lead to both delays in DNA replication and lowered replication fidelity. Understanding the processes of DNA replication and repair will require further investigation of the roles that the componentsof the replication machinery play in these processes in vivo. We thank NEELAMA M I N , BRADMERRII.~. and DAVIDROSE for work on new mutant alleles of the CDC44 gene; WILLIAM ROMANOWfor work on the mutation frequency analysis; BERNARD KUNZ for providing yeast strains and plasmids; MARTIN BUDDfor advice on sucrose gradients; and members of the HOLMlab for suggestions and com-

RFC in DNA Replication and Repair ments on this work. This work was supported by grant GM-36510 to C.H. from the National Institutes of Health.

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