Genetic Analyses of Schizosaccharomyces Pombe Dna2؉ Reveal That Dna2 Plays an Essential Role in Okazaki Fragment Metabolism

Genetic Analyses of Schizosaccharomyces pombe dna2؉ Reveal That Dna2 Plays an Essential Role in Okazaki Fragment Metabolism

Ho-Young Kang,*,† Eunjoo Choi,* Sung-Ho Bae,* Kyoung-Hwa Lee,* Byung-Soo Gim,* Hee-Dai Kim,* Chankyu Park,† Stuart A. MacNeill‡ and Yeon-Soo Seo* *National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Changan-Ku Suwon, Kyunggi-Do, 440-746, Korea, †Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusung-Ku, Taejon, 305-701, Korea and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom Manuscript received November 17, 1999 Accepted for publication March 31, 2000

ABSTRACT In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2ϩ of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further deﬁne its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2ϩ was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2ϩ led to chromosome fragmentation strikingly similar to that seen when cdc17ϩ, the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase ␦ (cdc1ϩ and cdc27ϩ), DNA ligase I (cdc17ϩ), and Fen-1 (rad2ϩ). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2ϩ plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2ϩ acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.

T the initiation of chromosomal DNA replication, lagging strands. Pol ␦ is involved in the elongation of A strand separation occurs to establish replication the RNA-DNA primers on the lagging strand template forks. Due to the antiparallel structure of double helix (Okazaki fragment elongation) as well as the replication DNA and the conserved 5Ј to 3Ј polarity of all DNA of the leading strand. Pol ␦ (and pol ε) requires two polymerases known to date, one strand (designated the accessory factors, PCNA and RFC, for its processive DNA leading strand) is continuously synthesized in the direc- synthesis. Saccharomyces cerevisiae pol ␦ complex is com- tion of fork movement. The other strand (the lagging posed of three subunits having apparent molecular strand) grows discontinuously in a direction opposite masses of 125, 58, and 55 kD encoded by the POL3, to fork movement (Kornberg and Baker 1992). The POL31, and POL32 genes, respectively (Gerik et al. generation of a continuous DNA strand from the short 1998). Studies of Schizosaccharomyces pombe have identi- and discontinuous lagging-strand fragment can be re- fied four subunits of pol ␦ that migrate with apparent garded as the most frequent and yet complex enzymatic molecular masses of 125, 55, 54, and 22 kD that are event at replication forks. encoded by pol3ϩ/cdc6ϩ, cdc1ϩ, cdc27ϩ, and cdm1ϩ, re- Okazaki fragment synthesis requires the action of poly- spectively (MacNeill et al. 1996; Zuo et al. 1997). merase (pol) ␣-primase, DNA pol ␦, and/or ε with pro- Okazaki fragments are ligated together through a proliferating nuclear antigen (PCNA) and replication fac- cess called Okazaki fragment maturation, which re- tor-C (RFC; Stillman 1994; Bambara et al. 1997; Baker quires the combined action of Fen-1 (also called 5Ј to and Bell 1998). Pol ␣, tightly complexed with DNA 3Ј exonuclease, MF1, or DNase IV), RNase HI, and DNA primase, plays a role in the initiation of DNA synthesis ligase I (Ishimi et al. 1988; Goulian et al. 1990; Waga by providing RNA-DNA primers for both leading and and Stillman 1994; Waga et al. 1994). In the current model, RNA primers are removed by Fen-1 (assisted by RNase HI), followed by gap filling by DNA pol ␦ (and/ Corresponding author: Yeon-Soo Seo, National Creative Research Ini- or pol ε) and the joining of the nicks by DNA ligase I. tiative Center for Cell Cycle Control, Samsung Biomedical Research Recently, it was shown that Fen-1 is a structure-specific Institute, Sungkyunkwan University School of Medicine, 300 Chun- chun-Dong, Changan-Ku Suwon, Kyunggi-Do, 440-746, Korea. endonuclease that cleaves at the junction of a flap struc- E-mail: [email protected] ture (Bambara et al. 1997; Lieber 1997). This suggests

Genetics 155: 1055–1067 ( July 2000) 1056 H.-Y. Kang et al. that branch structures may be generated during Okazaki the 5Ј primer oligoribonucleotides (S.-H. Bae and Y.-S. fragment metabolism. The mechanism, however, by Seo, unpublished results). In addition to a biochemical which the unannealed branch structure is generated is approach, we sought in vivo evidence for a role of DNA2 yet to be discovered. Moreover, the RAD27 gene (also in Okazaki fragment metabolism. For this purpose, we called RTH1) encoding S. cerevisiae Fen-1 (yFen-1 or isolated the S. pombe homolog (dna2ϩ)ofS. cerevisiae Rad27) is not essential in vivo, although cells lacking DNA2 and constructed ts alleles of dna2ϩ. Characteriza- RAD27 are inviable at certain growth conditions (e.g., tion of the S. pombe dna2 mutants revealed that S. pombe 37Њ; Reagan et al. 1995; Sommers et al. 1995). The RNase Dna2 interacted genetically with Cdc1 and Cdc2 (sub- HI gene (RNH35)inS. cerevisiae is not required for units of pol ␦), Rad2 (S. pombe homolog of yFen-1), and either DNA replication or cell growth (Frank et al. Cdc17 (DNA ligase I). All of these gene products are 1998). Instead, the deletion of RAD27 increased the essential for either elongation or maturation of Okazaki rates of spontaneous mutation, mitotic recombination, fragments. Our results extend the previous observations and chromosome loss (Johnson et al. 1995; Reagan et to another organism and present new in vivo data that al. 1995; Vallen and Cross 1995), consistent with it dna2ϩ is directly involved in Okazaki fragment metabo- having critical roles for chromosome maintenance lism. On the basis of our genetic studies, we propose (DeMott et al. 1996, 1998; Klungland and Lindahl a novel mechanism by which Dna2 participates as a 1997; Tishkoff et al. 1997; Freudenreich et al. 1998; component of a multienzyme complex for the synthesis Kim et al. 1998; Gary et al. 1999a; Wu et al. 1999). These and processing of Okazaki fragments. results deemphasize the only known role of Fen-1 for DNA replication and strongly argue for the existence MATERIALS AND METHODS of an alternative enzymatic system that allows cells to endure the loss of Fen-1/RNase HI functions. Strains and growth media: The following S. pombe strains Genetic studies in S. cerevisiae uncovered a component were used in this study (Table 1). The haploid strain HK100 Ϫ likely to be involved in Okazaki fragment maturation (h ura4-D18 leu1-32) was used to isolate the temperature- sensitive mutants. The diploid strain EC1 (hϩ/hϪ leu1-32/leu1- by virtue of its genetic and physical association with 32 ura4-D18/ura4-D18 ade6-M210/ade6-M216) was con- Fen-1 (Budd and Campbell 1997), adding further com- structed by mating ED666 (hϩ leu1-32 uraD-18 ade6-M210) and plexity to Okazaki fragment processing. The essential ED667 (hϪ leu1-32 uraD-18 ade6-M216) and was used for gene DNA2 gene of S. cerevisiae encodes a 172-kD protein with disruption (ED666 and ED667, gifts from Dr. J. Rho, Seoul Ϫ characteristic DNA helicase motifs (Budd and Camp- National University, Korea). The h haploid strains with either cdc17-k42 or cdc24-M38 (Nasmyth and Nurse 1981) and bell 1995; Budd et al. 1995). DNA2 homologs are found rad2::ura4ϩ alleles (gifts of Dr. J. Murray, University of Sussex, throughout eukaryotes including humans, plants, fish, UK) were used to evaluate the effects of combining mutations and nematodes (Budd and Campbell 1997; Formosa (synthetic lethality) with dna2-C2. The haploid strains carrying and Nittis 1999), suggesting that its role may be evolu- hus1-14 (Enoch et al. 1992) or rhp9::ura4ϩ (Willson et al. tionarily conserved in all eukaryotes. A specific associa- 1997; gifts from Dr. F. Z. Watts, University of Sussex, United Kingdom) were used to construct the dna2-C2 hus1-14 or dna2- tion of yFen-1 and S. cerevisiae Dna2 was demonstrated C2 rhp9::ura4ϩ strain, respectively. S. pombe cells were grown both genetically and biochemically (Budd and Camp- either in YE or Edinburgh minimal medium (EMM) media bell 1997). Cells harboring temperature-sensitive (ts) supplemented with appropriate nutrients (Alfa et al. 1993). alleles of S. cerevisiae DNA2 arrested at either G2/M Transformation of S. pombe was performed as described (Pren- with a 2C DNA content at the restrictive temperature tice 1992). For constructions of strains used in this study (Table 1), standard S. pombe genetic methods were used (Mor- (Fiorentino and Crabtree 1997) or at S phase (Budd eno et al. 1991). and Campbell 1995), depending on the Dna2 alleles DNA, oligonucleotides, plasmids, and libraries: All PCR used. It was also speculated that Dna2 displaces RNA primers or oligonucleotides used were commercially synthe- primers from template DNA by translocating along the sized (BioServe Biotechnologies, Laurel, MD). Primers A and template DNA as does pol ␦, creating a flap-like substrate B (degenerate primers; 5Ј-GGN-ATG-CCN-GGN-ACN-GGN- AAR-ACN-AC-3Ј and 5Ј-DAT-RTT-GTC-NAC-NGC-RCT-RTG- for Fen-1 endonuclease (Bambara et al. 1997; Waga NGT-RTA-3Ј, respectively) were used to amplify the dna2ϩ and Stillman 1998). This provided a possible mecha- gene fragment of S. pombe. Primers C and D (5Ј-CGG GAT nism by which Dna2 is involved in Okazaki fragment CCA TAT GGA TTT TCC AGG TCT G-3Ј and 5Ј-CCG CTC maturation. Recently, however, we showed that the re- GAG AAT TAA GCA AAC TAA GCT-3Ј, respectively) were ϩ combinant S. cerevisiae Dna2 protein intrinsically con- used to amplify cdc24 and primers E and F (5Ј-CGG GAT CCT TAT GCG AAC AGT ATT TTC G-3Ј and 5Ј-CCG CTC tained a strong single-stranded DNA-specific endonucle- GAG TCA GCA GTA ACT CTC AGC TA-3Ј, respectively) were ase activity (Bae et al. 1998), providing a possible used for cdc17ϩ. The primers G and H (5Ј-GAA TTC ATG function for Dna2 in Okazaki fragment maturation. Un- GAG GAA TGG AGA AAC TT-3Ј and 5Ј-CTC GAG TTA TTT fortunately, this process is poorly understood and re- CTT TCC AAA AAA GG-3Ј, respectively) were used to obtain ϩ mains to be defined more clearly. cdc27 . The 54-mer oligonucleotide (5Ј-AAG TAA GAA GTA TTT TCT TCT TTT TGG CAA GCA ATG ATC TGA TTA To test this possibility, we characterized the endonu- AGC TAG AAA-3Ј) contained the unique internal sequence clease activity of S. cerevisiae Dna2 and found that Dna2 of the amplified dna2ϩ fragment and was used as a probe to possessed many enzymatic activities capable of removing screen full-length cDNA or genomic DNA of dna2ϩ. Role of Dna2 in Okazaki Fragment Metabolism 1057

The genomic dna2ϩ gene was cloned into pBluescript TABLE 1 SK(ϩ) plasmid (Stratagene, La Jolla, CA) between the EcoRI and XhoI sites to make pSK-dna2ϩ. A 3.9-kb EcoRI-KpnI frag- Strains used in this study ment (Figure 1B, EcoRI in multiple cloning sites of vector and unique KpnI within dna2ϩ) and a 3.1-kb SalI-XhoI fragment Strain Genotype (Figure 1B, unique SalI within dna2ϩ and XhoI in multiple ϩ Ϫ cloning sites of the vector) from pSK-dna2ϩ were indepen- EC1 h /h leu1-32/leu1-32 ura4-D18/ura4-D18 ade6- dently cloned into pTZ19R (Pharmacia, Piscataway, NJ) to M210/ade6-M216 ϩ Ϫ ϩ construct pSpdna2N and pSpdna2C, respectively. A BamHI EC2 h /h dna2::ura4 /ϩ leu1-32/leu1-32 ura4-D18/ fragment (1.8 kb) from pTZ19R-cdc1EBg⌬U(MacNeill et ura4-D18 ade6-M210/ade6-M216 al. 1996) and a HindIII fragment (1.8 kb) from pGRP-130 (a EC3 hϩ/hϪ dna2ϩ/dna2ϩ leu1-32/leu1-32 ura4ϩ/ura4- plasmid harboring ura4ϩ gene flanked by HindIII sites) were D18 ade6-M210/ade6-M216 cloned into pSpdna2N and pSpdna2C, respectively, to intro- HK10 hϪ dna2-C2 leu1-32 ura4-D18 duce the ura4ϩ selection marker gene into the vectors. These HK11 hϪ dna2-C1 leu1-32 ura4-D18 two plasmids were designated pTZ-dna2N and pTZ-dna2C, HK12 hϪ dna2-C2 hus1-14 leu1-32 ura4-D18 respectively (Figure 1B) and used for random mutagenesis in HK13 hϪ dna2-C2 rhp9::ura4ϩ leu1-32 ura4-D18 vivo. A pUR19-based genomic library (Barbet et al. 1992; a HK14 hϩ cdc17-K42 leu1-32 gift from Dr. H. Ohkura, University of Edinburgh, United HK15 hϩ rad2::ura4ϩ leu1-32 aed6-704 ura4-D18 Kingdom) was used to screen for multicopy suppressors of HK16 hϩ cdc24-M38 dna2-C2 mutants. HK100 hϪ leu1-32 ura4-D18 To construct the plasmid pREP1-dna2ϩ in which dna2ϩ is placed under the control of the nmt1 promoter, full-length Except HK100, all strains were derived or constructed from dna2ϩ cDNA was cloned into the pBlueBacHis2 vector (In- the original sources described in materials and methods. vitrogen, Carlsbad, CA) between the BamHI and KpnI sites to create pBBH-dna2ϩ. In this vector, dna2ϩ cDNA is flanked by two EcoRI sites or the XhoI sites of the vector origin. The XhoI ϩ Temperature-sensitive dna2 mutants were screened and iso- fragment of dna2 was blunted by the use of Klenow and then lated using the strategy of Francesconi et al. (1993), except ligated into blunt-ended SalI sites of pREP1. that the gene was mutagenized by amplification of plasmids Cloning of cDNA and genomic DNA and characterization in an Escherichia coli mutator strain (Epicurian Coli XL 1-Red; of its transcript: Degenerate primers were designed from the Stratagene) deficient in three major DNA repair pathways. conserved regions between DNA2 of S. cerevisiae and its human The plasmids, pTZ-dna2N or pTZ-dna2C, were introduced homologue open reading frame (Eki et al. 1996). Degenerate into and amplified in the E. coli mutator strain. The plasmids primers A and B (200 pmol each) were used for amplification recovered were digested with NdeI (pTZ-dna2N) or with NcoI of the S. pombe genomic DNA template (600 ng) in 20 ␮lof (pTZ-dna2C; Figure 1B) and were used to transform the reaction buffer (Promega, Madison, WI). Four bands 350, HK100 strain to Uraϩ on EMM plates supplemented with 150, 110, and 75 bp in size were amplified after 12 cycles leucine at 25Њ.Uraϩ transformants had integrated the linear- (annealing, 1 min at 45Њ) plus 26 cycles (annealing, 1 min at ized DNA by homologous recombination at the dna2 locus to 50Њ) of PCR (extension, 1 min at 72Њ; denaturation, 1 min produce a complete dna2 gene and a truncated copy separated at 95Њ) in a thermocycler (MJ Research, Watertown, MA). by the ura4ϩ gene. Uraϩ colonies from the pTZ-dna2N and Sequencing analysis revealed that the 110-bp PCR-derived the pTZ-dna2C (9 ϫ 103 and 1 ϫ 104 transformants, respec- band contained the sequence that is highly conserved with tively) were replica plated on EMM supplemented with leucine S. cerevisiae DNA2 (Budd and Campbell 1995). The 54-mer and Phloxin B (1.75 ␮g/ml) and screened for clones that did oligonucleotide from the internal sequence of the 110-bp PCR not grow at the elevated temperature (37Њ). To recover the product was synthesized and used as a specific probe to screen integrated plasmids, the total DNA was isolated from clones a S. pombe cDNA library in pGAD-GH (Clontech, Palo Alto, that did not grow at the restrictive temperature. The DNA 5 CA). Among 1 ϫ 10 colonies screened, one cDNA clone of was digested with an appropriate restriction enzyme (e.g., NcoI a 970-bp insert was obtained. The insert was radiolabeled and for pTZ-dna2C integrants; Figure 1), ligated to recircularize used as a probe to further screen S. pombe cDNA and genomic the plasmids, and then introduced into E. coli DH5␣ strain. DNA libraries (gifts of Dr. H. Yoo, Korea Research Institute The plasmids were recovered from the resulting ampicillin- of Bioscience and Biotechnology) to isolate additional clones resistant transformants. The sequences of mutant alleles were ϩ containing the missing 5Ј and 3Ј terminus of dna2 . Full- also determined. -length cDNA and genomic DNA of dna2ϩ were cloned by Analyses of dna2::ura4؉ and dna2-C2 cells: An overnight cul repeating these procedures and their sequences were deter- ture (1 ml) of EC2 diploid strain (Table 1) was inoculated mined with an automated DNA sequencer (ABI PRISM 310 into EMM (200 ml) supplemented with leucine and glutamate Genetic Analyzer from Perkin-Elmer, Norwalk, CT). instead of NH4Cl as the nitrogen source and incubated at 30Њ Gene disruption and screening for temperature-sensitive for 72 hr with shaking. The cells were harvested and washed mutant alleles: The PstI fragment (Figure 1A, 0.8 kb, an inter- with 200 ml of sterile water and then resuspended in sterile nal region of dna2ϩ) of pSK-dna2ϩ was subcloned into pBlue- water (200 ml) containing 0.5 ml of Helix promatia juice (Sepra- Script SK(ϩ) to construct pSK-0.8PstI. The HindIII fragment cor, France) to digest ascus walls. After incubation at 30Њ for (1.8 kb, the intact ura4ϩ gene) was isolated from pREP2 (a 18 hr with shaking, the spores were harvested, washed with gift of Dr. J. Hurwitz at Sloan-Kettering Institute) and inserted 100 ml sterile water three times, and then resuspended in 10 into the unique HindIII site located within the subcloned PstI ml of sterile water. This suspension was inoculated into 200 fragment in pSK-0.8PstI. The resulting construct was digested ml of EMM supplemented with adenine and leucine (OD595 and the cultures were incubated with shaking at (0.15ف with PstI and introduced into the EC1 diploid strain by electro- of poration to obtain a dna2ϩ knockout strain. Stable Uraϩ trans- 30Њ. Samples (10 ml, 100 ␮l) were taken every 3 hr for flow formants were isolated and verified for integration of the cytometry and cell number determination, respectively. Sam- marker gene at the desired locus by PCR and genomic South- ples for DAPI (4Ј,6-diamidino-2-phenylindole) staining were ern analyses. also taken at 19 hr after inoculation. As a control, wild-type 1058 H.-Y. Kang et al. dna2ϩ spores were identically prepared using diploid strain EC3 (Table 1), which is heterozygous for ura4ϩ (ura4ϩ/ura4- D18), in which half of the spores produced were dna2ϩ and uracil prototrophic spores. The analyses of dna2-C2 mutants were also performed as described above for dna2ϩ-deleted spores except that the culture was grown in EMM supplemented with leucine and uracil and shifted to 37Њ for the indicated times before sampling. Flow cytometry analysis and DAPI staining were performed as described (MacNeill and Fantes 1994). Screening for multicopy suppressors of dna2-C2 mutant: The HK10 haploid strain (Table 1) was grown at 25Њ to midlog phase in EMM supplemented with leucine and uracil. The genomic library in pUR19 was transformed into dna2-C2 mutants, and the cells were plated on EMM plates containing leucine and were allowed to grow at 25Њ for 24 hr and at 34.5Њ for an additional 4–6 days. The temperature-tolerant Uraϩ transformants were selected and streaked on EMM plates containing leucine at 25Њ. The plasmids were recovered from the ϩ candidate Ura transformants and checked for their ability Figure 1.—Structure of the dna2ϩ gene and positions of to suppress the ts phenotype by reintroducing them into dna2- dna2 ts mutations. (A) The dna2ϩ exons are indicated by four C2 mutants. The sequences were then determined and ana- open bars, and the three introns are denoted by thin lines lyzed using the BLAST server (http://www.ncbi.nlm.nih.gov/ (intron 1, nucleotide positions 359–403; intron 2, 3198–3246; cgi-bin/BLAST/nph-newblast). intron 3, 3962–4008). The numbers 1 and 4334 on the scale Yeast two-hybrid assays: The EcoRI fragment from pBBH- bar above the dna2ϩ gene refer to the adenine of the start dna2ϩ as described above was inserted into pGBT9 (Clontech) ϩ codon (AUG) and the last nucleotide of the stop codon for GAL4 DNA-binding domain fusion. The cdc24 cDNA was (UGA), respectively. For the construction of dna2ϩ-disrupted amplified from an S. pombe cDNA library (a gift of Dr. H. Yoo, mutants, the PstI fragment (indicated by two wedges above Korea Research Institute of Bioscience and Biotechnology) the second exon) was replaced by the ura4ϩ gene as described using primers C and D (complementary to the 5Ј- and 3Ј-end ϩ in materials and methods. The positions of dna2-C1 (C to of cdc24 and containing BamHI and XhoI sites, respectively). T; Pro to Leu) and dna2-C2 (T to C; Leu to Ser) are The amplified cdc24ϩ cDNA was directly cloned into pCR2.1 956 1079 ϩ denoted by asterisks above the open reading frame. The five TA cloning vector (Invitrogen) to construct pCR2.1-cdc24 ; solid bars within exons and roman numerals below stand for DNA sequencing was carried out to assure that there was no the conserved helicase motifs from 21 related proteins (Hodg- erroneous nucleotide inserted in cdc24ϩ cDNA during PCR ϩ man 1988). The two shaded bars, designated by lowercase amplification. The BamHI-XhoI fragment (1.5 kb) of cdc24 roman numerals (i and ii) in the second exon, indicate con- cDNA was then inserted into pGAD424 between BamHI and served amino acid sequences in the N-terminal half among SalI (compatible with XhoI) sites to make pGAD424-cdc24ϩ. ϩ three Dna2 homologs from humans, budding yeast, and fission The PCR amplification of cdc17 cDNA (using primers E and yeast. (B) Two thick lines represent the fragments used to F) and the construction of pGAD424-cdc17ϩ were carried out construct plasmids pTZ-dna2N and pTZ-dna2C for mutagene- using the same strategy as for cdc24ϩ. Using primers G and sis of the dna2ϩ gene to obtain conditionally lethal mutants. H, pGAD424-cdc27ϩ containing cdc27ϩ cDNA was also made The arrows indicate the enzyme sites used to construct ts using the same strategy for cdc24ϩ except that the 5Ј primer mutants (NdeI, nucleotide position 1728; SalI, 1852; NcoI, 2569; (primer G) contained an EcoRI site instead of BamHI. The and KpnI, 2719). (C) The amino acid sequences of conserved BamHI restriction fragment from pET28c-cdc1ϩ (a gift from helicase motifs in S. pombe Dna2 are presented using the single- Dr. J. Hurwitz at Sloan-Kettering Institute) was cloned into letter code. The identical amino acid sequences conserved the BamHI site of pGAD424 to construct pGAD424-cdc1ϩ. The among the three Dna2 ORFs from humans and two yeasts are pACT2-rad2ϩ plasmids were obtained from Dr. J. Murray (Uni- versity of Sussex, United Kingdom). S. cerevisiae Y190 strain was shown as boldface capital letters and their positions in S.pombe Dna2 are indicated in parentheses. The DNA sequence of transformed using the lithium acetate method as described ϩ (Gietz et al. 1992) and Leuϩ Trpϩ colonies were selected on dna2 was deposited in GenBank as accession no. AF144384. SD plates. The transformants were transferred to Whatman filter papers (cat. no. 1005 090) presoaked with Z buffer (Miller 1972) containing ␤-mercaptoethanol (38.6 mm) and cloned by PCR amplification and repeated cycles of X-gal (0.33 mg/ml). The filter papers were frozen at Ϫ70Њ or in liquid nitrogen and thawed at room temperature to standard screening procedures as described in materi- permeabilize the cells. The filter papers were then placed als and methods. Both genomic and cDNA sequences on another presoaked filter and incubated at 30Њ until the of dna2ϩ have been deposited into GenBank under ac- appearance of blue color. The colonies exhibiting positive cession no. AF144384. Alignment of nucleotide se- blue colors were then picked from original plates and re- quences from genomic and cDNA clones showed that streaked on SD plates. The ␤-galactosidase assays were performed using liquid cultures as described (Miller 1972; Kai- the open reading frame was interrupted by three introns ser et al. 1994). [nucleotide positions starting from adenine (ϩ1) of the initiation codon, 359–403; 3198–3246; and 3962–4008; Figure 1A). Computer analysis identified a single open RESULTS reading frame (ORF) of 4191 nucleotides that encoded Isolation and structure of the dna2؉ gene: The dna2ϩ a 158-kD protein with 1397 amino acids. In support of gene, an S. pombe homolog of budding yeast DNA2, was this, we detected the 4.6-kb mRNA transcript by North- Role of Dna2 in Okazaki Fragment Metabolism 1059 ern blot analysis (not shown). While this study was in progress, an identical gene was isolated as a multicopy suppressor of the cdc24-G1 ts mutant and named dna2ϩ on the basis of its significant homology with S. cerevisiae DNA2 (Gould et al. 1998). Recently, the complete sequence of the dna2ϩ gene was released from the Sanger Center (GenBank accession no. CAB38508), but contained an ORF one amino acid longer than the one we cloned. The extra amino acid reported by the Sanger Center might result from a computer prediction that chose the first splicing acceptor site in the first intron among two possible candidate acceptor sites, adding one amino acid at position 120. The S. pombe Dna2 protein contains conserved helicase motifs I, II, III, V, and VI, characteristic of helicases (Hodgman 1988; Gorbalenya et al. 1989); these motifs are localized to the C-terminal one-third of the protein (Figure 1, A and C). The dna2؉ gene product is essential, but not required for initiation and elongation stages of DNA replication during germination of spores: We investigated whether S. pombe dna2ϩ is essential for cell viability by disrupting dna2ϩ using S. pombe strain EC1 (Table 1), as described in materials and methods. A dna2::ura4ϩ/dna2ϩ heterozygous diploid strain (EC2, Table 1) was sporulated on malt extract agar (ME) plates. Tetrad analyses of the resulting asci reproducibly yielded two viable spores, both of which were UraϪ (9 out of 10 tetrads tested; 1 tetrad showed only one viable UraϪ spore), indicating that dna2ϩ is an essential gene like S. cerevisiae DNA2 (Budd and Campbell 1995; not shown). We examined the phenotype of the dna2ϩ-disrupted spores obtained from the heterozygous EC2 diploid strain. Spores formed from EC2 were inoculated into EMM lacking uracil. Under this growth condition, only spores pos- sessing the disrupted dna2::ura4ϩ gene could germinate and grow. Mutant cells taken at the 19-hr time point were stained with DAPI and examined microscopically Figure 2.—The dna2ϩ gene is not required for bulk DNA for their morphology (Figure 2A). Most dna2::ura4ϩ synthesis when cells are germinating or growing vegetatively. ϩ cells at this stage were highly elongated and mononu- (A) Wild-type and dna2 -disrupted cells were stained with DAPI and examined with fluorescence microscopy. (B) Flow clear, in keeping with a typical cell division cycle (cdc) cytometric analysis of wild-type and dna2ϩ-disrupted spores mutant phenotype (Figure 2A). during germination. Wild-type dna2ϩ/dna2ϩ diploid cells het- Since the Dna2 protein in S. cerevisiae was suspected erozygous for ura4ϩ (ura4ϩ/ura4-D18) as a positive control to have an important role in DNA replication (Budd were identically processed along with dna2ϩ/dna2::ura4ϩ dip- and Campbell 1995, 1997; Budd et al. 1995), we exam- loid cells. Spores obtained from both diploid strains germi- ϩ nated for 6 hr, after which their DNA content was determined ined the changes in DNA content of the dna2 -deleted by flow cytometry as described in materials and methods. spores during germination. In this experiment, wild- The positions of two DNA peaks (unreplicated and fully repli- type dna2ϩ/dna2ϩ diploid cells (EC3, Table 1) heterozy- cated) are indicated as 1C and 2C. (C) Wild-type and dna2- gous for ura4ϩ (ura4ϩ/ura4-D18) were used as a positive C2 cells incubated at 37Њ for 6 and 8 hr were stained with control and processed identically along with EC2 dip- DAPI and examined under fluorescence microscopy. loid cells. Spores obtained from both diploid strains were allowed to germinate for 6 hr, after which time samples were taken every 3 hr up to 18 hr. The cells pleted by 15 hr. Like wild type, the mutant spores were stained with propidium iodide for flow cytometry (dna2::ura4ϩ) were capable of initiating DNA replica- analyses to investigate their DNA content. As shown in tion, but their rate of replication was slower and com- Figure 2B (left), DNA replication of wild-type spores pleted 18 hr after inoculation (Figure 2B, right). (dna2ϩ) was initiated 6–9 hr after inoculation and com- Isolation and characterization of temperature-sensi- 1060 H.-Y. Kang et al. tive mutants: To examine the effect of mutations of dna2ϩ in vegetatively growing cells, we constructed conditional mutants of dna2ϩ and investigated their phenotype. Two putative dna2 ts mutants were obtained from cells (HK100) transformed with a linearized plasmid pTZ-dna2C that had been in vivo mutagenized (Figure 1, A and B). The verification that these mutants were ts was carried out as follows (not shown, unless indicated otherwise): (i) The ura4ϩ marker was stably maintained and tightly linked to the temperature lethality; (ii) the plasmid, pREP1-dna2ϩ containing the wild-type cDNA of dna2ϩ, was able to rescue the ts mutants (Figure 5; in addition, the plasmid pTZ-dna2C that was not subjected to mutagenic treatment was able to abolish the ts phenotype when integrated into the chromosome of candidate mutants); and (iii) plasmids recovered Figure 3.—Cells carrying a dna2-C2 mutant allele are sensi- from the putative mutants reestablished the ts pheno- tive to alkylating agents and a DNA replication inhibitor. Cells type when introduced into wild-type cells after being with either wild-type (wt) or mutant (dna2-C2, designated by C2) alleles of dna2ϩ were grown on minimal medium con- linearized. The two ts mutants isolated satisfied all of taining the indicated concentration of drugs at the permissive these criteria, establishing that they contained muta- temperature (28Њ). The number of wild-type or mutant cells tions associated specifically with the chromosomal was first determined, and then serially diluted samples (104, 3 2 dna2ϩ gene. These two ts mutants were named dna2-C1 10 ,10, and 10 cells) were spotted and grown for 4 days and dna2-C2 (Figure 1A). The mutations were C-G to on plates containing the indicated concentrations of drugs, methyl methanesulfonate (MMS) or hydroxyurea (HU). T-A (dna2-C1) and T-A to C-G (dna2-C2) transitions, resulting in the alteration of amino acid residue Pro956 to Leu and Leu1079 to Ser, respectively. The two residues ture of 28Њ, which does not affect the growth of the

Pro956 and Leu1079 are conserved from budding yeast to dna2-C2 mutant (Figure 3). The dna2-C2 mutant was human, and Pro956 is located in the nucleotide-binding sensitive to methylmethane sulfonate (MMS) and motif (Figure 1A). At the permissive temperature, cells slightly sensitive to 10 mm hydroxyurea (HU) but not carrying the dna2-C1 allele showed a slight growth de- to UV (doses ranging from 0 to 400 J/m2) compared fect, whereas those with the dna2-C2 allele showed no to wild type (Figure 3). In view of its remarkable sensitiv- differences in growth, compared to wild-type cells (not ity to MMS, an alkylating agent, dna2ϩ is likely to play shown). Following a shift to the nonpermissive tempera- an important role in DNA repair, although the mecha- ture (37Њ for 6–8 hr), dna2-C2 cells arrested as elongated nism by which this occurs is unclear. The HK11 strain cells with a single nucleus (Figure 2C) that doubled containing the dna2-C1 allele (Table 1) responded simi- their DNA content (measured by FACScan analyses, not larly to the various genotoxic agents tested above, like shown). Cells carrying dna2-C1, subjected to the same the dna2-C2 mutant (not shown). analysis, yielded identical results (not shown). These The absence of dna2؉ function triggers the replication findings are strikingly similar to those obtained with checkpoint: To test whether dna2ϩ is involved in DNA dna2::ura4ϩ disrupted spores (Figure 2A) and indicate replication, we constructed a strain containing both that bulk chromosomal DNA replication occurs in the dna2-C2 and hus1-14 mutations. The hus1ϩ gene plays a absence of a functional dna2ϩ product. These results role in the DNA replication checkpoint: hus1-14 mutant are in accordance with those obtained for S. cerevisiae cells with unreplicated DNA or damaged DNA fail to DNA2 (Fiorentino and Crabtree 1997), suggesting arrest at G2 and proceed into mitosis with fatal conse- that the in vivo function of Dna2 is conserved in the quences (Enoch et al. 1992). Thus, if dna2ϩ is required two yeasts. We concluded that the dna2ϩ gene of S. for DNA replication, the introduction of the checkpoint pombe is not required for the initiation and elongation mutation, hus1-14, into cells containing the dna2-C2 mu- of replication forks, essential steps to replicate the chro- tation should allow the double mutant cells to enter mosomal DNA en masse, regardless of modes of growth. mitosis, leading to catastrophic events at the nonpermis- The temperature-sensitive mutation causes impaired sive temperature. Indeed, a significant percentage resistance to distinct genotoxic agents: Since inactiva- (Ͼ11%) of the hus1-14 dna2-C2 double mutant cells tion of the dna2ϩ gene product did not affect DNA (HK12, Table 1) underwent aberrant mitosis when synthesis, we investigated the effects of genotoxic agents shifted to the nonpermissive temperature (37Њ), produc- on HK10 dna2-C2 mutant strain (Table 1) in an effort ing anucleated cells or progeny cells in which DNA to gain insight into the role(s) played by the dna2ϩ was distributed unevenly (Figure 4A, right). In contrast, gene in DNA transactions other than replication. The such aberrant mitosis was not detected in control cells experiments were done at the semipermissive tempera- containing the hus1-14 mutation alone (Figure 4A, left) Role of Dna2 in Okazaki Fragment Metabolism 1061 or a dna2-C2 single mutant, which arrested as shown in Figure 2. The same catastrophic result was obtained when HK13 cells (Table 1) containing both dna2-C2 and rhp9::ura4ϩ were examined (not shown). The rhp9ϩ gene, a fission yeast homolog of S. cerevisiae RAD9,is required for the DNA damage checkpoint, but not for the replication checkpoint (Weinert and Hartwell 1988; Willson et al. 1997). These results suggest that the dna2-C2 mutant at 37Њ most likely results in the incomplete replication of chromosomal DNA, which probably generates damaged DNA structures recog- nized by the DNA damage checkpoint. This suggests that nicks or ssDNA regions are present in the newly replicated DNA in the absence of dna2ϩ. Therefore, we conclude that the dna2ϩ gene has an essential function in DNA replication at a stage leading to the completion of duplex DNA synthesis. -Loss of dna2؉ function causes qualitatively incom plete chromosome replication: The results described above indicate that the absence of dna2ϩ function leads to a defect in DNA replication. To further confirm this, we analyzed the structure of S. pombe chromosomes using pulsed-field gel electrophoresis (PFGE). As shown in Figure 4B, chromosomes from wild-type cells entered the gel and were separated into three chromosomes irrespective of the incubation temperature (Figure 4B, lanes 1–4). However, chromosomes isolated from dna2- C2 mutant cells (HK10) that were incubated Ͼ4hrat Figure 4.—Loss of dna2ϩ function triggers checkpoint con- the nonpermissive temperature failed to yield separated trol and results in chromosome breakage. (A) The control chromosomes (Figure 4B, lanes 7 and 8). Interestingly, strain (hus1-14) or the double mutant HK12 strain containing hus1-14 dna2-C2 was grown at 25Њ and shifted to 37Њ for 8 hr. the low molecular weight smear of DNA observed in The cells were then stained with DAPI and examined under the dna2-C2 mutant was similar to that found in cdc17- a microscope. Note that cells on the right were enlarged to K42 cells (HK14) whose wild-type protein, DNA ligase observe the “cut” phenotypes (arrows) more closely. The dou- I, functions in the maturation of Okazaki fragments ble mutant cells contained heavily fragmented DNAs, which (Johnston et al. 1986; Waga et al. 1994). The frag- appear as speckled spots or unevenly distributed DNA. (B) Wild-type, HK10 (dna2-C2), and HK14 (cdc17-K42) cells grown mented DNA in both cases accumulated at the bottom in EMM supplemented with leucine and uracil at 25Њ were of the gel (Figure 4B). In addition, similarly fragmented shifted to 37Њ. The cells were further incubated for 0 (lanes DNA was also observed in cdc24 mutants whose growth 1, 5, and 9), 2 (lanes 2, 6, and 10), 4 (lanes 3, 7, and 11), defect was suppressed by dna2ϩ (Gould et al. 1998). and 8 (lanes 4, 8, and 12) hr at 37Њ. Agarose plugs were The smear of DNA may result from a qualitative defect prepared from these cells and the chromosomes were resolved by pulsed-field gel electrophoresis (PFGE) as described of DNA replication such as pausing of replication forks (Gould et al. 1998). Each plug contained 108 cells and PFGE exposing frail single-stranded DNA or generation of was carried out in 0.6% agarose in a CHEF-DR III apparatus premature Okazaki fragments that have nicks or an un- (Bio-Rad, Richmond, CA) for 72 hr in 0.5ϫ TAE buffer at 1.5 processed flap structure. Thus, the resulting DNA be- V/cm with a switch time of 30 min and 120Њ of included angle. comes prone to breakage by nuclease attack or fragile In the case where the hydroxyurea arrest control was carried out (indicated as HU; lane 13), 12 mm (final concentration) during experimental manipulation. It is also worthwhile was added to wild-type cultures grown at 25Њ and cells were to mention that the S. cerevisiae dna2-1 mutant synthe- further incubated for 4 hr at 30Њ prior to preparation of the sized only low molecular weight DNA at the nonpermis- agarose plug. sive temperature in metabolic labeling studies, suggesting a defect similar to that of the S. pombe dna2-C2 mutant (Budd et al. 1995). These results, as well as those temperature sensitivity of the dna2-C2 mutant using the described above, indicate that the dna2-C2 mutation procedures described in materials and methods. The has a defect in DNA replication despite its ability to screening of 6.2 ϫ 105 HK10 cells transformed with the synthesize bulk DNA. genomic library yielded 47 independent transformants Isolation of multicopy suppressors for the dna2-C2 ts that grew at the restrictive temperature. Among these contained (%90ف) mutant: An S. pombe genomic DNA library in the pUR19 candidate suppressors, 42 clones vector was screened for genes that could rescue the wild-type dna2ϩ as expected, and five clones contained 1062 H.-Y. Kang et al.

Figure 5.—Temperature sensitivity of dna2 mutants is rescued by multicopy or overexpression of genes involved in Okazaki fragment elongation and maturation. (A) The mutant cells (HK10) carrying the dna2-C2 allele were transformed with either vector (pUR19) alone or pUR19 containing dna2ϩ, cdc27ϩ,orcdc17ϩ. Aliquots containing 104,103,102, and 10 transformed cells were spotted onto EMM plates containing leucine at the nonpermissive temperature of 34Њ. (B) The dna2-C2 mutant cells harboring vector (pREP3X) alone, pREP1-dna2ϩ, pREP3X-cdc1ϩ, or pREP41X-rad2ϩ under control of the nmt1 promoter were spotted (104,103,102, and 10 cells) and grown at 34Њ in the presence (ϩthi) or absence (Ϫthi) of thiamine on EMM plates containing uracil. Note that the mutants containing dna2ϩ grow faster in the presence of thiamine (repressed condition) than in the absence of thiamine because overexpression of dna2ϩ caused growth retardation (Formosa and Nittis 1999; Parenteau and Wellinger 1999). potential extragenic suppressors. Three clones had the pressed the growth defect of dna2-C2 weakly (cdm1ϩ, complete sequence of the cdc17ϩ gene encoding DNA encoding the 22-kD subunit of pol ␦) or not at all (pol3ϩ, ligase I (Johnston et al. 1986) in common. Two other encoding the catalytic subunit of pol ␦; not shown). clones contained both the complete sequence of the The rad2ϩ gene rescued the dna2-C2 defect when its putative S. pombe fas gene encoding the folic acid synthe- expression was induced in the absence of thiamine (Fig- sis protein (Volpe et al. 1992) and the 5Ј two-thirds of ure 5B). These results demonstrate that the genes in- the cdc27ϩ gene, a 54-kD subunit of the pol ␦ complex volved in the Okazaki fragment elongation or matura- (MacNeill et al. 1996). The latter two clones were ana- tion genetically interact with dna2ϩ. lyzed further to confirm which gene was responsible for The dna2-C2 mutation is synthetically lethal with cdc17- suppression. We found that cdc27ϩ alone was sufficient K42, rad2::ura4؉, and cdc24-M38: The data presented and necessary for the suppression of dna2-C2 mutation above support a role for dna2ϩ in the metabolism of (not shown). The growth of the dna2-C2 mutant con- Okazaki fragments. To further strengthen this conclu- taining either cdc17ϩ or cdc27ϩ (the 5Ј two-thirds partial sion, we examined whether the defect of dna2-C2 can sequence) as a multicopy suppressor is shown in Figure be exaggerated when combined with mutant alleles of 5A. In addition, the dna2-C1 mutation was also sup- genes such as cdc17-K42 (DNA ligase I) and rad2::ura4ϩ pressed equally well by either cdc17ϩ or cdc27ϩ (not (Murray et al. 1994). Tetrads obtained from the cross shown), suggesting that the defects associated with dna2- of dna2-C2 mutant cells (HK10) with either the cdc17- C1 and dna2-C2 are similar. K42 (HK14) or rad2::ura4ϩ (HK15) mutant were dis- Since cdc27ϩ and cdc17ϩ genes are required for the sected, and spores were grown at the permissive temper- elongation and maturation, respectively, of Okazaki ature of 25Њ (Table 2). In each case, a high proportion of fragments (Ishimi et al. 1988; Goulian et al. 1990; Waga dead spores was observed (Table 2; 29 and 21% inviable and Stillman 1994; Waga et al. 1994) and can suppress spores from 21 tetrads of HK10 ϫ HK14 and 14 tetrads the defect observed with the two dna2 ts mutations, it of HK10 ϫ HK15 crosses, respectively). The percentage is most likely that dna2ϩ functions in the Okazaki frag- of dead spores from the two crosses was close to the ment metabolism. For this reason, we investigated expected percentage of dead spores (25%), assuming whether other genes involved in Okazaki fragment me- the combination of the two unlinked mutations is lethal. tabolism also suppressed the temperature sensitivity of The dna2ϩ gene is on chromosome 2 and thus not the dna2-C2 mutation. We constructed plasmids that linked to cdc17ϩ, cdc24ϩ, and rad2ϩ, which are on chro- contained pol3ϩ, cdc1ϩ,orcdm1ϩ (pol ␦ subunits) or mosome 1. In test crosses between dna2-C2 and cdc17- rad2ϩ (an S. pombe homolog of RAD27) under the con- K42 or between dna2-C2 and rad2::ura4ϩ, no double trol of the nmt1 promoter (Forsburg 1993; Maundrell mutants were detected among the growing spores, con- 1993). The wild-type dna2ϩ rescued the temperature- firming that dna2-C2 is synthetically lethal when com- sensitive lethality regardless of the presence or absence bined with cdc17-K42 or rad2::ura4ϩ (Table 2). of thiamine (Figure 5B). This is due to the high basal We also examined the genetic interaction between level activity of nmt1 promoter in pREP1 (Maundrell dna2ϩ and cdc24ϩ, a novel replication gene of fission 1993), which is sufficient to complement the dna2-C2 yeast essential for chromosome integrity (Gould et al. ts mutation. The dna2-C2 mutant cells did not grow at all 1998), to determine whether these two genes are syn- when the expression of cdc1ϩ or rad2ϩ was not induced thetically lethal. For this purpose, we crossed dna2-C2 (Figure 5B). The induction of cdc1ϩ in the absence (HK10) and cdc24-M38 (HK16) mutant strains and ana- of thiamine allowed the dna2-C2 ts mutants to grow lyzed the resulting tetrads. Among 15 tetrads dissected, efficiently at the restrictive temperature (Figure 5B). In 25% of the total spores obtained were inviable at 25Њ contrast, induction of the other pol ␦ subunits sup- (Table 2) and no double mutant was found, suggesting Role of Dna2 in Okazaki Fragment Metabolism 1063

TABLE 2 Mutations caused synthetic lethality when combined with dna2-C2

Tetrad typesa dna2-C2 Total Viable Inviable combined with PD TT NPD spores sporesb spores (%) cdc17-K42 4 10 7 84 60 29 rad2::ura4ϩ 563 564421 cdc24-M38 393 604525 The dna2-C2 mutant was crossed with cells carrying cdc17-k42, rad2::ura4ϩ,orcdc24-M38 and resulting tetrads were dissected and grown at 25Њ. PD, parental ditype; TT, tetratype; NPD, nonparental ditype. a The tetrad types were determined by examining the phenotype of each spore and we excluded from the analyses the tetrads containing spores that did not germinate. Briefly, in PD tetrads obtained from the cross of dna2-C2 ϫ cdc17-K42, all the progeny spores were viable and showed a temperature-sensitive (ts) phenotype. In NPD tetrads, two spores were wild type (temperature tolerant phenotype) and two spores were inviable although they germinated under microscopic examination. In TT tetrads, one out of four spores was inviable and the remaining three viable spores gave a 2:1 segregation of ts:wild type. The absence of double ts mutations in viable spores was confirmed by backcrossing ts spores with wild-type cells. The backcross always gave rise to a 2:2 segregation of ts:wild type. The cross of dna2-C2 ϫ cdc24-M38 or dna2-C2 ϫ rad2::ura4ϩ was similarly analyzed. b The viability of spores from control crosses was normally Ͼ89%. a synthetic lethal interaction between the two genes. transformed with pGBT9-dna2ϩ and pGAD424-cdc24ϩ Fifteen tetrads from crossing of dna2-C2 with cdc27-P11 turned blue within 1 hr when assayed for ␤-galactosidase (MacNeill et al. 1996) were also analyzed. Unlike the in filter assays (see materials and methods), whereas others above, dna2-C2 cdc27-P11 double mutants were control cells hardly turned blue even after 36 hr. This recovered at the expected one-in-four frequency and result indicates a strong interaction between S. pombe viable at both 25Њ (permissive temperature) and 30Њ Dna2 and Cdc24. We observed that the reciprocal inter- (semipermissive temperature). The failure to observe action using pGBT9-cdc24ϩ (BD fusion) and pGAD424- a synthetic lethal interaction between the two mutant dna2ϩ (AD fusion) was weaker, but still significant (not alleles may be due to allele specificity. If, for example, shown). When pGBT9-dna2ϩ was cotransformed with a specific physical interaction is important for viability, either pGAD424-cdc1ϩ or pACT2-rad2ϩ (AD fusions), only those mutant alleles that do not allow the physical reduced levels of ␤-galactosidase activity were detected interaction would cause the double mutants to be invia- (Table 3). Although these activities were relatively low, fold higher than controls-10ف ble. The synthetic lethality of dna2-C2 when combined they were reproducibly with mutant alleles of several genes known to function in with either pGBT9-dna2ϩ, pGAD424-cdc1ϩ, or pACT2- elongation or maturation of Okazaki fragments suggests rad2ϩ alone (Table 3 and not shown). This result sug- that dna2ϩ is involved in DNA replication, especially at gests weak, but meaningful, interaction between the two the stage of Okazaki fragment metabolism. proteins. In keeping with this, cells cotransformed with S. pombe Dna2 interacts with Cdc24, Cdc1, and Rad2 pGBT9-dna2ϩ/pGAD424-cdc1ϩ and pGBT9-dna2ϩ/ in the yeast two-hybrid system: Since we confirmed the pACT2-rad2ϩ turned blue within 8 hr in filter assays. genetic interactions of dna2ϩ with cdc24ϩ, rad2ϩ, cdc27ϩ, In contrast, cells containing pGBT9-dna2ϩ, pGAD424- cdc1ϩ, and cdc17ϩ, we decided to investigate the physical cdc1ϩ, or pACT2-rad2ϩ each alone did not develop blue interactions between dna2ϩ and those genes using the S. color at Ͼ36 hr. The reciprocal combinations failed to cerevisiae two-hybrid assay. We constructed a bait plasmid lead to detectable ␤-galactosidase activity (not shown), (pGBT9-dna2ϩ) containing dna2ϩ fused to the GAL4 suggesting orientation-specific interactions in the two- DNA-binding domain (BD) in pGBT9. The cdc24ϩ, hybrid assay. When pGBT9-dna2ϩ was cotransformed rad2ϩ, cdc27ϩ, cdc1ϩ, and cdc17ϩ genes were fused to the with either pGAD424-cdc27ϩ or pGAD424-cdc17ϩ, the GAL4 activation domain (AD) in pGAD424 or pACT2 to resulting transformants failed to show ␤-galactosidase prepare prey plasmids (pGAD424-cdc24ϩ, pACT2-rad2ϩ, activity above background levels (Table 3). However, pGAD424-cdc27ϩ, pGAD424-cdc1ϩ, and pGAD424- the cotransformed cells developed a pale blue color cdc17ϩ, respectively) as described in materials and after prolonged incubation (Ͼ18 hr) in filter assays, times more sensitive than the liquid assay 106ف methods. The plasmid pGBT-dna2ϩ alone or in combi- which are nation with pGAD424 or each plasmid expressing a (Table 3). These observations were highly reproducible prey protein did not activate transcription of reporter and the control plasmid alone did not develop blue genes (Table 3 and not shown). When pGBT9-dna2ϩ color even after Ͼ36 h of incubation (not shown). (BD fusion) and pGAD424-cdc24ϩ (AD fusion) were To further confirm the interactions observed above, cotransformed, a high level of ␤-galactosidase activity we examined the expression of the HIS3 reporter gene was detected (Table 3). Consistent with this, cells co- by growing cells in the presence of various concentra- 1064 H.-Y. Kang et al.

TABLE 3 Summary of results from two-hybrid analyses

Interactions Baita Preyb ␤-galc X-gald 3-ATe Suppressionf dna2ϩ Vector ϪϪϪ dna2ϩ cdc24ϩ 38.9 ϩϩϩ ϩϩ ND dna2ϩ cdc1ϩ 0.30 ϩϩ Yes dna2ϩ cdc27ϩ ϪϮϪ Yes dna2ϩ cdc17ϩ ϪϮϪ Yes dna2ϩ rad2ϩ 0.38 ϩϩ Yes a The dna2ϩ was fused to the GAL4 DNA binding domain fusion. b The genes were fused to the GAL4 activation domain. c The interactions were determined by measuring ␤-galactosidase activities in Miller units using ONPG as a substrate (Miller 1972) and the values given represent the average of Figure 6.—The dna2ϩ interacts with cdc24ϩ, rad2ϩ, and Ϫ duplicate cultures. Background levels (indicated as )of cdc1ϩ in the yeast two-hybrid system. The budding yeast strain ␤ -galactosidase activity detected with bait or prey alone were Y190 harboring pGBT9-dna2ϩ as bait was transformed with Ͻ0.0041 Ϯ 0.004. ϩ ϩ d pGAD424 (vector), pGAD424-cdc24 , pGAD424-rad2 , and Interactions were monitored by the appearance of blue pGAD424-cdc1ϩ and the resulting transformants were exam- ϩϩϩ ϩ color ( , formation of blue color within 1 hr; , within ined for their ability to express the HIS3 reporter gene in the Ϯ Ͼ Ϫ Ͼ 6–8 hr; , 18 hr; , 36 hr) using the colony lift assay on presence of increasing concentrations of 3-aminotriazole (3- filter papers containing X-gal (0.33 mg/ml). AT). The cells (104,103,102, and 10) were spotted on synthetic e ϩϩ The HIS3 reporter gene was used to assess the growth ( , minimal medium lacking l-tryptophan, l-leucine, and l-histi- ϩ Ϫ wild-type-like growth; , moderate growth; , poor or no dine and containing 3-AT at the concentrations indicated. growth) on SD plates containing the highest concentration (30 mm) of 3-aminotriazole (see also Figure 6). f Suppression of dna2-C2 mutants by multicopy suppressors (cdc27ϩ and cdc17ϩ) or by overexpression of the genes under and cdc17ϩ, marginally interacted with dna2ϩ; their in- the control of nmt1 promoter (cdc1ϩ and rad2ϩ). ND, not teractions were detectable only in filter assays (Table determined. 3). In conclusion, the specific genetic and two-hybrid interactions of dna2ϩ with genes known (DNA ligase, Fen-1, and pol ␦) or implicated (Cdc24) in Okazaki tions of 3-aminotriazole (3-AT), which suppresses the fragment metabolism place dna2ϩ as a protein that plays leaky growth of false-positive cells (Mangus et al. 1998). a critical role in Okazaki fragment elongation/maturation. Cells containing each plasmid were grown to midlog phase and aliquots were spotted on SD plates containing increasing concentrations of 3-AT (Figure 6). The cells DISCUSSION containing either vector (pGBT9, Figure 6) or bait alone In this article, we showed that S. pombe spores or vege- ϩ (pGBT9-dna2 , not shown) failed to grow at high levels tative cells with an inactivated dna2ϩ exhibited a distinct of 3-AT (Figure 6). However, cells containing pGBT9- terminally arrested shape similar to that observed with ϩ ϩ ϩ dna2 plus pGAD424-cdc24 , pACT2-rad2 ,or the mutant cells defective in DNA replication genes ϩ pGAD424-cdc1 grew efficiently in the presence of up such as cdc24ϩ, cdc27ϩ,orpcn1ϩ (Waseem et al. 1992; to 30 mm 3-AT (Figure 6). At 20 mm 3-AT, the growth MacNeill et al. 1996; Gould et al. 1998). They doubled difference was indistinguishable in cells expressing their DNA content, however, suggesting that S. pombe ϩ ϩ ϩ cdc24 , rad2 , and cdc1 , whereas the growth of control Dna2 is not essential for the initiation of DNA replica- cells containing vector alone was extremely poor. These tion or the progression of replication forks as suggested results suggest that physical interactions exist between previously for S. cerevisiae Dna2 (Fiorentino and S. pombe Dna2 and Cdc24, Rad2, and Cdc1. We also Crabtree 1997). Since the precise role of a protein in investigated the interaction between the prey proteins, a multienzyme process such as DNA replication can be Cdc24, Rad2, Cdc27, and Cdc17, but did not detect suggested by the proteins with which it interacts, we any significant interaction among these proteins (not attempted to find those that can interact either geneti- shown), suggesting that S. pombe Dna2 serves as a scaffold cally or physically with S. pombe Dna2. We concluded protein capable of interacting simultaneously with sev- that S. pombe dna2ϩ plays a crucial role in the completion eral proteins. The interactions observed in the yeast of DNA synthesis on the basis of the following results: (i) two-hybrid system are consistent with the abilities of inactivation of S. pombe Dna2 triggered the replication cdc1ϩ, cdc27ϩ, cdc17ϩ, and rad2ϩ to suppress the temper- checkpoint; (ii) loss of S. pombe dna2ϩ function caused ature lethality of dna2-C2. Two of these genes, cdc27ϩ qualitatively incomplete chromosome replication (chro- Role of Dna2 in Okazaki Fragment Metabolism 1065 mosomes from the S. pombe dna2 mutant were heavily The growth of mutant cells containing rad27-n (a nu- fragmented at the nonpermissive temperature); and clease-deficient allele of RAD27) was severely inhibited, (iii) S. pombe Dna2 interacted genetically with Rad2, whereas the growth of rad27⌬ or rad27-p (defective in DNA ligase I, and two subunits of pol ␦ (Cdc1 and the PCNA-binding site) mutant cells was not (Gary et Cdc27), all of which are involved directly in Okazaki al. 1999a,b). The double mutant (rad27-n,p) was not fragment metabolism (Waga and Stillman 1998). inhibited. These observations were interpreted as fol- These findings in vivo are also consistent with the results lows. In rad27-n cells, the interaction of Rad27-n with from our studies in vitro showing that S. cerevisiae Dna2 PCNA allows the mutant protein to occupy its normal is a single-stranded DNA-specific endonuclease that is position within a multiprotein complex, which could well suited to completely remove primer RNA on Oka- hinder the action of secondary or redundant enzymes zaki fragments (Bae et al. 1998; S.-H. Bae and Y.-S. Seo, capable of processing the Okazaki fragment. In the case unpublished results). of rad27-n,p or rad27-p, the mutant proteins were not We observed an increased sensitivity of dna2-C2 mu- integrated within the multiprotein complex, hence tants to MMS. Since MMS creates adducts and apurinic allowing access of an alternative enzyme. These observa- sites, which become single- and double-strand breaks tions not only support the idea that processing of Oka- that result from a failure to replicate past lesions con- zaki fragments occurs in the context of a multiprotein taining 3-methyladenine (Schwartz 1989), the dna2 assembly, but also predict the existence of a secondary mutants used in this study most likely lack the ability to processing enzyme that can operate in the absence of remove damaged DNA prior to replicating the lesions, Fen-1 (Rad27). We believe that the enzyme is most likely suggesting a role for Dna2 in DNA repair. The S. pombe Dna2 for the reasons described elsewhere (S.-H. Bae mutant dna2 alleles that were isolated mapped inside and Y.-S. Seo, unpublished results). the helicase domain (Figure 1), in keeping with the Dna2 may be regulated by proteins with which it inter- previous observation that MMS-sensitive alleles of S. cere- acts. Such a possibility is supported by significant differ- visiae DNA2 clustered in the helicase domain, and the ences in enzymatic activities noted between the recombi- site-directed motif I and motif II mutations caused MMS nant S. cerevisiae Dna2 purified from insect cells and the sensitivity (Formosa and Nittis 1999). This result sup- one from S. cerevisiae cell extracts (Budd and Campbell ports the notion that the helicase function is needed 1995; Bae et al. 1998). Alternatively, Dna2 can alter for some forms of DNA damage repair. enzymatic properties of proteins such as pol ␦ or Fen-1 The ability of S. pombe dna2ϩ to interact with two via protein-protein interaction in order to coordinate subunits of pol ␦, Rad2, and DNA ligase I raises the the complicated multienzyme process of Okazaki frag- possibility that Dna2 exists in vivo within a multiprotein ment elongation and maturation. For example, the spe- complex. On the basis of the results from yeast two- cific interaction between Dna2 and pol ␦ would lead to hybrid analyses (Table 3), however, interactions of S. a change in the ability of pol ␦ to displace the 5Ј-end pombe Dna2 with two subunits (Cdc1 and Cdc27) of pol region of the preexisting Okazaki fragments. We showed ␦ may not be strong enough to allow formation of a that a flap structure generated by displacement DNA stable complex between Dna2 and pol ␦. Other interac- synthesis by pol ␦ was rapidly removed by S. cerevisiae tions of S. pombe Dna2 with Rad2 and DNA ligase I would Dna2 (S.-H. Bae and Y.-S. Seo, unpublished results). be necessary for Dna2 to be stably tethered to pol ␦. Considering that pol ␦ is capable of displacement DNA Under these circumstances, the ability of Rad2 and DNA synthesis up to 274 bp, longer than the size (100–150 ligase I to complex directly with PCNA (Li et al. 1995; nucleotides) of Okazaki fragments (Mossi et al. 1998), Levin et al. 1997; Montecucco et al. 1998; Gary et al. one possible consequence of Dna2-pol ␦ interaction 1999b) could stabilize further the association of S. pombe would be the timely disassembly of the pol ␦ complex Dna2 with pol ␦, since PCNA itself is tightly coupled to when no further displacement is required. At present, pol ␦ while DNA synthesis occurs. This would account the roles played by Dna2 in the context of a multienzyme for the following discrepancy: we failed to observe any complex are highly conjectural and rigorous biochemi- detectable complex formed between purified recombi- cal studies are needed to define any role of Dna2 in nant Rad27 and Dna2 of S. cerevisiae (not shown). In this regard. Recently, an additional role for Dna2 has contrast, both Rad27 and S. cerevisiae Dna2 copurified on been suggested by the observation that S. cerevisiae Dna2 an immunoaffinity column, and they were reciprocally interacts with POL1 and CTF4, which encode the DNA coimmunoprecipitated from crude extracts (Budd and polymerase ␣ catalytic subunit and an associated pro- Campbell 1997). In support of the hypothesis that Dna2 tein, respectively (Formosa and Nittis 1999). This sug- exists in a multiprotein complex, S. cerevisiae Dna2 in gests that Dna2 may also act in a process that involves cell extracts eluted from a size-exclusion matrix column pol ␣ in addition to the roles suggested above. Although -kD, although this complex has not been ana- pol ␣-primase plays an essential role in Okazaki frag 700ف at lyzed yet (Formosa and Nittis 1999). ment initiation (Tsurimoto et al. 1990; Waga and Recent genetic studies on the mutant alleles of rad27 Stillman 1994; Waga et al. 1994), the precise link be- are also in accord with our hypothesis (Gary et al. 1999b). tween Dna2, pol ␦, and pol ␣ is not clearly understood. 1066 H.-Y. Kang et al.

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Watts (University of Sussex, UK), Dr. H. Ohkura Gary, R., M. S. Park, J. P. Nolan, H. L. Cornelius, O. G. Kozyreva (University of Edinburgh, UK), Dr. J. Hurwitz (Sloan-Kettering Insti- et al., 1999b A novel role in DNA metabolism for the binding of Fen1/Rad27 to PCNA and implications for genetic risk. Mol. tute, USA), Dr. J. Rho (Seoul National University, Korea), and Dr. Cell. Biol. 19: 5373–5382. H. Yoo (Korea Research Institute of Bioscience and Biotechnology, Gerik, K. J., X. Li, A. Pautz and P. M. J. Burgers, 1998 Characteriza- Korea). We are greatly indebted to A. Sanderson (University of Edin- tion of the two small subunits of Saccharomyces cerevisiae DNA burgh, UK) for FACScan analyses. This work was supported by a polymerase delta. J. Biol. Chem. 273: 19747–19755. grant from the Creative Research Initiatives of the Korean Ministry Gietz, D., A. St. Jean, R. A. Woods and R. H. 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