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

Corresponding author: Yeon-Soo Seo, National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchun-Dong, Changan-Ku Suwon, Kyunggi-Do, 440-746, Korea., ysseo{at}medical.skku.ac.kr (E-mail)

Communicating editor: G. R. SMITH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2⁺ 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 dna2⁺ was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2⁺ led to chromosome fragmentation strikingly similar to that seen when cdc17⁺, 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 (cdc1⁺ and cdc27⁺), DNA ligase I (cdc17⁺), and Fen-1 (rad2⁺). 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 dna2⁺ plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2⁺ acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.

AT the initiation of chromosomal DNA replication, strand separation occurs to establish replication forks. Due to the antiparallel structure of double helix DNA and the conserved 5' to 3' polarity of all DNA polymerases known to date, one strand (designated the leading strand) is continuously synthesized in the direction of fork movement. The other strand (the lagging strand) grows discontinuously in a direction opposite to fork movement (KORNBERG and BAKER 1992 ). The generation of a continuous DNA strand from the short and discontinuous lagging-strand fragment can be regarded as the most frequent and yet complex enzymatic event at replication forks.

Okazaki fragment synthesis requires the action of polymerase (pol) -primase, DNA pol , and/or with proliferating nuclear antigen (PCNA) and replication factor-C (RFC; STILLMAN 1994 ; BAMBARA et al. 1997 ; BAKER and BELL 1998 ). Pol , tightly complexed with DNA primase, plays a role in the initiation of DNA synthesis by providing RNA-DNA primers for both leading and lagging strands. Pol is involved in the elongation of the RNA-DNA primers on the lagging strand template (Okazaki fragment elongation) as well as the replication of the leading strand. Pol (and pol ) requires two accessory factors, PCNA and RFC, for its processive DNA synthesis. Saccharomyces cerevisiae pol complex is composed of three subunits having apparent molecular masses of 125, 58, and 55 kD encoded by the POL3, POL31, and POL32 genes, respectively (GERIK et al. 1998 ). Studies of Schizosaccharomyces pombe have identified four subunits of pol that migrate with apparent molecular masses of 125, 55, 54, and 22 kD that are encoded by pol3⁺/cdc6⁺, cdc1⁺, cdc27⁺, and cdm1⁺, respectively (MACNEILL et al. 1996 ; ZUO et al. 1997 ).

Okazaki fragments are ligated together through a process called Okazaki fragment maturation, which requires the combined action of Fen-1 (also called 5' to 3' exonuclease, MF1, or DNase IV), RNase HI, and DNA ligase I (ISHIMI et al. 1988 ; GOULIAN et al. 1990 ; WAGA 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/or pol ) and the joining of the nicks by DNA ligase I. Recently, it was shown that Fen-1 is a structure-specific endonuclease that cleaves at the junction of a flap structure (BAMBARA et al. 1997 ; LIEBER 1997 ). This suggests that branch structures may be generated during Okazaki fragment metabolism. The mechanism, however, by which the unannealed branch structure is generated is yet to be discovered. Moreover, the RAD27 gene (also called RTH1) encoding S. cerevisiae Fen-1 (yFen-1 or Rad27) is not essential in vivo, although cells lacking RAD27 are inviable at certain growth conditions (e.g., 37°; REAGAN et al. 1995 ; SOMMERS et al. 1995 ). The RNase HI gene (RNH35) in S. cerevisiae is not required for either DNA replication or cell growth (FRANK et al. 1998 ). Instead, the deletion of RAD27 increased the rates of spontaneous mutation, mitotic recombination, and chromosome loss (JOHNSON et al. 1995 ; REAGAN et al. 1995 ; VALLEN and CROSS 1995 ), consistent with it having critical roles for chromosome maintenance (DEMOTT et al. 1996 , DEMOTT et al. 1998 ; KLUNGLAND and LINDAHL 1997 ; TISHKOFF et al. 1997 ; FREUDENREICH et al. 1998 ; KIM et al. 1998 ; GARY et al. 1999A ; WU et al. 1999 ). These results deemphasize the only known role of Fen-1 for DNA replication and strongly argue for the existence of an alternative enzymatic system that allows cells to endure the loss of Fen-1/RNase HI functions.

Genetic studies in S. cerevisiae uncovered a component likely to be involved in Okazaki fragment maturation by virtue of its genetic and physical association with Fen-1 (BUDD and CAMPBELL 1997 ), adding further complexity to Okazaki fragment processing. The essential DNA2 gene of S. cerevisiae encodes a 172-kD protein with characteristic DNA helicase motifs (BUDD and CAMPBELL 1995 ; BUDD et al. 1995 ). DNA2 homologs are found throughout eukaryotes including humans, plants, fish, and nematodes (BUDD and CAMPBELL 1997 ; FORMOSA and NITTIS 1999 ), suggesting that its role may be evolutionarily conserved in all eukaryotes. A specific association of yFen-1 and S. cerevisiae Dna2 was demonstrated both genetically and biochemically (BUDD and CAMPBELL 1997 ). Cells harboring temperature-sensitive (ts) alleles of S. cerevisiae DNA2 arrested at either G2/M with a 2C DNA content at the restrictive temperature (FIORENTINO and CRABTREE 1997 ) or at S phase (BUDD and CAMPBELL 1995 ), depending on the Dna2 alleles used. It was also speculated that Dna2 displaces RNA primers from template DNA by translocating along the template DNA as does pol , creating a flap-like substrate for Fen-1 endonuclease (BAMBARA et al. 1997 ; WAGA and STILLMAN 1998 ). This provided a possible mechanism by which Dna2 is involved in Okazaki fragment maturation. Recently, however, we showed that the recombinant S. cerevisiae Dna2 protein intrinsically contained a strong single-stranded DNA-specific endonuclease activity (BAE et al. 1998 ), providing a possible function for Dna2 in Okazaki fragment maturation. Unfortunately, this process is poorly understood and remains to be defined more clearly.

To test this possibility, we characterized the endonuclease activity of S. cerevisiae Dna2 and found that Dna2 possessed many enzymatic activities capable of removing the 5' primer oligoribonucleotides (S.-H. BAE and Y.-S. SEO, unpublished results). In addition to a biochemical approach, we sought in vivo evidence for a role of DNA2 in Okazaki fragment metabolism. For this purpose, we isolated the S. pombe homolog (dna2⁺) of S. cerevisiae DNA2 and constructed ts alleles of dna2⁺. Characterization of the S. pombe dna2 mutants revealed that S. pombe Dna2 interacted genetically with Cdc1 and Cdc2 (subunits of pol ), Rad2 (S. pombe homolog of yFen-1), and Cdc17 (DNA ligase I). All of these gene products are essential for either elongation or maturation of Okazaki fragments. Our results extend the previous observations to another organism and present new in vivo data that dna2⁺ is directly involved in Okazaki fragment metabolism. On the basis of our genetic studies, we propose a novel mechanism by which Dna2 participates as a component of a multienzyme complex for the synthesis and processing of Okazaki fragments.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and growth media:
The following S. pombe strains were used in this study (Table 1). The haploid strain HK100 (h^- ura4-D18 leu1-32) was used to isolate the temperature-sensitive mutants. The diploid strain EC1 (h⁺/h^- leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-M210/ade6-M216) was constructed by mating ED666 (h⁺ leu1-32 uraD-18 ade6-M210) and ED667 (h^- leu1-32 uraD-18 ade6-M216) and was used for gene disruption (ED666 and ED667, gifts from Dr. J. Rho, Seoul National University, Korea). The h^- haploid strains with either cdc17-k42 or cdc24-M38 (NASMYTH and NURSE 1981 ) and rad2::ura4⁺ alleles (gifts of Dr. J. Murray, University of Sussex, UK) were used to evaluate the effects of combining mutations (synthetic lethality) with dna2-C2. The haploid strains carrying hus1-14 (ENOCH et al. 1992 ) or rhp9::ura4⁺ (WILLSON et al. 1997 ; gifts from Dr. F. Z. Watts, University of Sussex, United Kingdom) were used to construct the dna2-C2 hus1-14 or dna2-C2 rhp9::ura4⁺ strain, respectively. S. pombe cells were grown either in YE or Edinburgh minimal medium (EMM) media supplemented with appropriate nutrients (ALFA et al. 1993 ). Transformation of S. pombe was performed as described (PRENTICE 1992 ). For constructions of strains used in this study (Table 1), standard S. pombe genetic methods were used (MORENO et al. 1991 ).

DNA, oligonucleotides, plasmids, and libraries:
All PCR primers or oligonucleotides used were commercially synthesized (BioServe Biotechnologies, Laurel, MD). Primers A and B (degenerate primers; 5'-GGN-ATG-CCN-GGN-ACN-GGN-AAR-ACN-AC-3' and 5'-DAT-RTT-GTC-NAC-NGC-RCT-RTG-NGT-RTA-3', respectively) were used to amplify the dna2⁺ gene fragment of S. pombe. Primers C and D (5'-CGG GAT CCA TAT GGA TTT TCC AGG TCT G-3' and 5'-CCG CTC GAG AAT TAA GCA AAC TAA GCT-3', respectively) were used to amplify cdc24⁺ and primers E and F (5'-CGG GAT CCT TAT GCG AAC AGT ATT TTC G-3' and 5'-CCG CTC GAG TCA GCA GTA ACT CTC AGC TA-3', respectively) were used for cdc17⁺. The primers G and H (5'-GAA TTC ATG GAG GAA TGG AGA AAC TT-3' and 5'-CTC GAG TTA TTT CTT TCC AAA AAA GG-3', respectively) were used to obtain cdc27⁺. The 54-mer oligonucleotide (5'-AAG TAA GAA GTA TTT TCT TCT TTT TGG CAA GCA ATG ATC TGA TTA AGC TAG AAA-3') contained the unique internal sequence of the amplified dna2⁺ fragment and was used as a probe to screen full-length cDNA or genomic DNA of dna2⁺.

The genomic dna2⁺ gene was cloned into pBluescript SK(+) plasmid (Stratagene, La Jolla, CA) between the EcoRI and XhoI sites to make pSK-dna2⁺. A 3.9-kb EcoRI-KpnI fragment (Fig 1B, EcoRI in multiple cloning sites of vector and unique KpnI within dna2⁺) and a 3.1-kb SalI-XhoI fragment (Fig 1B, unique SalI within dna2⁺ and XhoI in multiple cloning sites of the vector) from pSK-dna2⁺ were independently cloned into pTZ19R (Pharmacia, Piscataway, NJ) to construct pSpdna2N and pSpdna2C, respectively. A BamHI fragment (1.8 kb) from pTZ19R-cdc1EBgU (MACNEILL et al. 1996 ) and a HindIII fragment (1.8 kb) from pGRP-130 (a plasmid harboring ura4⁺ gene flanked by HindIII sites) were cloned into pSpdna2N and pSpdna2C, respectively, to introduce the ura4⁺ selection marker gene into the vectors. These two plasmids were designated pTZ-dna2N and pTZ-dna2C, respectively (Fig 1B) and used for random mutagenesis in vivo. A pUR19-based genomic library (BARBET et al. 1992 ; a gift from Dr. H. Ohkura, University of Edinburgh, United Kingdom) was used to screen for multicopy suppressors of dna2-C2 mutants.

View larger version (17K): In this window In a new window Download PPT slide   Figure 1. Structure of the dna2+ gene and positions of dna2 ts mutations. (A) The dna2+ exons are indicated by four open bars, and the three introns are denoted by thin lines (intron 1, nucleotide positions 359–403; intron 2, 3198–3246; intron 3, 3962–4008). The numbers 1 and 4334 on the scale bar above the dna2+ gene refer to the adenine of the start codon (AUG) and the last nucleotide of the stop codon (UGA), respectively. For the construction of dna2+-disrupted mutants, the PstI fragment (indicated by two wedges above the second exon) was replaced by the ura4+ gene as described in MATERIALS AND METHODS. The positions of dna2-C1 (C to T; Pro956 to Leu) and dna2-C2 (T to C; Leu1079 to Ser) are denoted by asterisks above the open reading frame. The five solid bars within exons and roman numerals below stand for the conserved helicase motifs from 21 related proteins (HODGMAN 1988 ). The two shaded bars, designated by lowercase roman numerals (i and ii) in the second exon, indicate conserved amino acid sequences in the N-terminal half among three Dna2 homologs from humans, budding yeast, and fission yeast. (B) Two thick lines represent the fragments used to construct plasmids pTZ-dna2N and pTZ-dna2C for mutagenesis of the dna2+ gene to obtain conditionally lethal mutants. The arrows indicate the enzyme sites used to construct ts mutants (NdeI, nucleotide position 1728; SalI, 1852; NcoI, 2569; and KpnI, 2719). (C) The amino acid sequences of conserved helicase motifs in S. pombe Dna2 are presented using the single-letter code. The identical amino acid sequences conserved among the three Dna2 ORFs from humans and two yeasts are shown as boldface capital letters and their positions in S.pombe Dna2 are indicated in parentheses. The DNA sequence of dna2+ was deposited in GenBank as accession no. AF144384.

To construct the plasmid pREP1-dna2⁺ in which dna2⁺ is placed under the control of the nmt1 promoter, full-length dna2⁺ cDNA was cloned into the pBlueBacHis2 vector (Invitrogen, Carlsbad, CA) between the BamHI and KpnI sites to create pBBH-dna2⁺. In this vector, dna2⁺ cDNA is flanked by two EcoRI sites or the XhoI sites of the vector origin. The XhoI fragment of dna2⁺ was blunted by the use of Klenow and then ligated into blunt-ended SalI sites of pREP1.

Cloning of cDNA and genomic DNA and characterization of its transcript:
Degenerate primers were designed from the conserved regions between DNA2 of S. cerevisiae and its human homologue open reading frame (EKI et al. 1996 ). Degenerate primers A and B (200 pmol each) were used for amplification of the S. pombe genomic DNA template (600 ng) in 20 µl of reaction buffer (Promega, Madison, WI). Four bands 350, 150, 110, and 75 bp in size were amplified after 12 cycles (annealing, 1 min at 45°) plus 26 cycles (annealing, 1 min at 50°) of PCR (extension, 1 min at 72°; denaturation, 1 min at 95°) in a thermocycler (MJ Research, Watertown, MA). Sequencing analysis revealed that the 110-bp PCR-derived band contained the sequence that is highly conserved with S. cerevisiae DNA2 (BUDD and CAMPBELL 1995 ). The 54-mer oligonucleotide from the internal sequence of the 110-bp PCR product was synthesized and used as a specific probe to screen a S. pombe cDNA library in pGAD-GH (Clontech, Palo Alto, CA). Among 1 x 10⁵ colonies screened, one cDNA clone of a 970-bp insert was obtained. The insert was radiolabeled and used as a probe to further screen S. pombe cDNA and genomic DNA libraries (gifts of Dr. H. Yoo, Korea Research Institute of Bioscience and Biotechnology) to isolate additional clones containing the missing 5' and 3' terminus of dna2⁺. Full-length cDNA and genomic DNA of dna2⁺ were cloned by repeating these procedures and their sequences were determined with an automated DNA sequencer (ABI PRISM 310 Genetic Analyzer from Perkin-Elmer, Norwalk, CT).

Gene disruption and screening for temperature-sensitive mutant alleles:
The PstI fragment (Fig 1A, 0.8 kb, an internal region of dna2⁺) of pSK-dna2⁺ was subcloned into pBlue-Script SK(+) to construct pSK-0.8PstI. The HindIII fragment (1.8 kb, the intact ura4⁺ gene) was isolated from pREP2 (a gift of Dr. J. Hurwitz at Sloan-Kettering Institute) and inserted into the unique HindIII site located within the subcloned PstI fragment in pSK-0.8PstI. The resulting construct was digested with PstI and introduced into the EC1 diploid strain by electroporation to obtain a dna2⁺ knockout strain. Stable Ura⁺ transformants were isolated and verified for integration of the marker gene at the desired locus by PCR and genomic Southern analyses.

Temperature-sensitive dna2 mutants were screened and isolated using the strategy of FRANCESCONI et al. 1993 , except that the gene was mutagenized by amplification of plasmids in an Escherichia coli mutator strain (Epicurian Coli XL 1-Red; Stratagene) deficient in three major DNA repair pathways. The plasmids, pTZ-dna2N or pTZ-dna2C, were introduced into and amplified in the E. coli mutator strain. The plasmids recovered were digested with NdeI (pTZ-dna2N) or with NcoI (pTZ-dna2C; Fig 1B) and were used to transform the HK100 strain to Ura⁺ on EMM plates supplemented with leucine at 25°. Ura⁺ transformants had integrated the linearized DNA by homologous recombination at the dna2 locus to produce a complete dna2 gene and a truncated copy separated by the ura4⁺ gene. Ura⁺ colonies from the pTZ-dna2N and the pTZ-dna2C (9 x 10³ and 1 x 10⁴ transformants, respectively) were replica plated on EMM supplemented with leucine and Phloxin B (1.75 µg/ml) and screened for clones that did not grow at the elevated temperature (37°). To recover the integrated plasmids, the total DNA was isolated from clones that did not grow at the restrictive temperature. The DNA was digested with an appropriate restriction enzyme (e.g., NcoI for pTZ-dna2C integrants; Fig 1), ligated to recircularize the plasmids, and then introduced into E. coli DH5 strain. The plasmids were recovered from the resulting ampicillin-resistant transformants. The sequences of mutant alleles were also determined.

Analyses of dna2::ura4⁺ and dna2-C2 cells:
An overnight culture (1 ml) of EC2 diploid strain (Table 1) was inoculated into EMM (200 ml) supplemented with leucine and glutamate instead of NH₄Cl as the nitrogen source and incubated at 30° for 72 hr with shaking. The cells were harvested and washed with 200 ml of sterile water and then resuspended in sterile water (200 ml) containing 0.5 ml of Helix promatia juice (Sepracor, France) to digest ascus walls. After incubation at 30° for 18 hr with shaking, the spores were harvested, washed with 100 ml sterile water three times, and then resuspended in 10 ml of sterile water. This suspension was inoculated into 200 ml of EMM supplemented with adenine and leucine (OD₅₉₅ of ~0.15) and the cultures were incubated with shaking at 30°. Samples (10 ml, 100 µl) were taken every 3 hr for flow cytometry and cell number determination, respectively. Samples for DAPI (4',6-diamidino-2-phenylindole) staining were also taken at 19 hr after inoculation. As a control, wild-type dna2⁺ spores were identically prepared using diploid strain EC3 (Table 1), which is heterozygous for ura4⁺ (ura4⁺/ura4-D18), in which half of the spores produced were dna2⁺ and uracil prototrophic spores. The analyses of dna2-C2 mutants were also performed as described above for dna2⁺-deleted spores except that the culture was grown in EMM supplemented with leucine and uracil and shifted to 37° 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 25° 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 25° for 24 hr and at 34.5° for an additional 4–6 days. The temperature-tolerant Ura⁺ transformants were selected and streaked on EMM plates containing leucine at 25°. The plasmids were recovered from the candidate Ura⁺ transformants and checked for their ability to suppress the ts phenotype by reintroducing them into dna2-C2 mutants. The sequences were then determined and analyzed using the BLAST server (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast).

Yeast two-hybrid assays:
The EcoRI fragment from pBBH-dna2⁺ as described above was inserted into pGBT9 (Clontech) for GAL4 DNA-binding domain fusion. The cdc24⁺ cDNA was amplified from an S. pombe cDNA library (a gift of Dr. H. Yoo, Korea Research Institute of Bioscience and Biotechnology) using primers C and D (complementary to the 5'- and 3'-end of cdc24⁺ and containing BamHI and XhoI sites, respectively). The amplified cdc24⁺ cDNA was directly cloned into pCR2.1 TA cloning vector (Invitrogen) to construct pCR2.1-cdc24⁺; DNA sequencing was carried out to assure that there was no erroneous nucleotide inserted in cdc24⁺ cDNA during PCR amplification. The BamHI-XhoI fragment (1.5 kb) of cdc24⁺ cDNA was then inserted into pGAD424 between BamHI and SalI (compatible with XhoI) sites to make pGAD424-cdc24⁺. The PCR amplification of cdc17⁺ cDNA (using primers E and F) and the construction of pGAD424-cdc17⁺ were carried out using the same strategy as for cdc24⁺. Using primers G and H, pGAD424-cdc27⁺ containing cdc27⁺ cDNA was also made using the same strategy for cdc24⁺ except that the 5' primer (primer G) contained an EcoRI site instead of BamHI. The BamHI restriction fragment from pET28c-cdc1⁺ (a gift from Dr. J. Hurwitz at Sloan-Kettering Institute) was cloned into the BamHI site of pGAD424 to construct pGAD424-cdc1⁺. The pACT2-rad2⁺ plasmids were obtained from Dr. J. Murray (University of Sussex, United Kingdom). S. cerevisiae Y190 strain was transformed using the lithium acetate method as described (GIETZ et al. 1992 ) and Leu⁺ Trp⁺ colonies were selected on SD plates. The transformants were transferred to Whatman filter papers (cat. no. 1005 090) presoaked with Z buffer (MILLER 1972 ) containing ß-mercaptoethanol (38.6 mM) and X-gal (0.33 mg/ml). The filter papers were frozen at -70° or in liquid nitrogen and thawed at room temperature to permeabilize the cells. The filter papers were then placed on another presoaked filter and incubated at 30° until the appearance of blue color. The colonies exhibiting positive blue colors were then picked from original plates and restreaked on SD plates. The ß-galactosidase assays were performed using liquid cultures as described (MILLER 1972 ; KAISER et al. 1994 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and structure of the dna2⁺ gene:
The dna2⁺ gene, an S. pombe homolog of budding yeast DNA2, was cloned by PCR amplification and repeated cycles of standard screening procedures as described in MATERIALS AND METHODS. Both genomic and cDNA sequences of dna2⁺ have been deposited into GenBank under accession no. AF144384. Alignment of nucleotide sequences from genomic and cDNA clones showed that the open reading frame was interrupted by three introns [nucleotide positions starting from adenine (+1) of the initiation codon, 359–403; 3198–3246; and 3962–4008; Fig 1A). Computer analysis identified a single open reading frame (ORF) of 4191 nucleotides that encoded a 158-kD protein with 1397 amino acids. In support of this, we detected the 4.6-kb mRNA transcript by Northern 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 dna2⁺ on the basis of its significant homology with S. cerevisiae DNA2 (GOULD et al. 1998 ). Recently, the complete sequence of the dna2⁺ 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 (Fig 1A and Fig 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 dna2⁺ is essential for cell viability by disrupting dna2⁺ using S. pombe strain EC1 (Table 1), as described in MATERIALS AND METHODS. A dna2::ura4⁺/dna2⁺ 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 Ura^- (9 out of 10 tetrads tested; 1 tetrad showed only one viable Ura^- spore), indicating that dna2⁺ is an essential gene like S. cerevisiae DNA2 (BUDD and CAMPBELL 1995 ; not shown). We examined the phenotype of the dna2⁺-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 possessing the disrupted dna2::ura4⁺ gene could germinate and grow. Mutant cells taken at the 19-hr time point were stained with DAPI and examined microscopically for their morphology (Fig 2A). Most dna2::ura4⁺ cells at this stage were highly elongated and mononuclear, in keeping with a typical cell division cycle (cdc) mutant phenotype (Fig 2A).

View larger version (42K): In this window In a new window Download PPT slide   Figure 2. The dna2+ gene is not required for bulk DNA synthesis when cells are germinating or growing vegetatively. (A) Wild-type and dna2+-disrupted cells were stained with DAPI and examined with fluorescence microscopy. (B) Flow cytometric analysis of wild-type and dna2+-disrupted spores during germination. Wild-type dna2+/dna2+ diploid cells heterozygous for ura4+ (ura4+/ura4-D18) as a positive control were identically processed along with dna2+/dna2::ura4+ diploid cells. Spores obtained from both diploid strains germinated for 6 hr, after which their DNA content was determined by flow cytometry as described in MATERIALS AND METHODS. The positions of two DNA peaks (unreplicated and fully replicated) are indicated as 1C and 2C. (C) Wild-type and dna2-C2 cells incubated at 37° for 6 and 8 hr were stained with DAPI and examined under fluorescence microscopy.

Since the Dna2 protein in S. cerevisiae was suspected to have an important role in DNA replication (BUDD and CAMPBELL 1995 , BUDD and CAMPBELL 1997 ; BUDD et al. 1995 ), we examined the changes in DNA content of the dna2⁺-deleted spores during germination. In this experiment, wild-type dna2⁺/dna2⁺ diploid cells (EC3, Table 1) heterozygous for ura4⁺ (ura4⁺/ura4-D18) were used as a positive control and processed identically along with EC2 diploid 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 were stained with propidium iodide for flow cytometry analyses to investigate their DNA content. As shown in Fig 2B (left), DNA replication of wild-type spores (dna2⁺) was initiated 6–9 hr after inoculation and completed by 15 hr. Like wild type, the mutant spores (dna2::ura4⁺) were capable of initiating DNA replication, but their rate of replication was slower and completed 18 hr after inoculation (Fig 2B, right).

Isolation and characterization of temperature-sensitive mutants:
To examine the effect of mutations of dna2⁺ in vegetatively growing cells, we constructed conditional mutants of dna2⁺ 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 (Fig 1A and Fig B). The verification that these mutants were ts was carried out as follows (not shown, unless indicated otherwise): (i) The ura4⁺ marker was stably maintained and tightly linked to the temperature lethality; (ii) the plasmid, pREP1-dna2⁺ containing the wild-type cDNA of dna2⁺, was able to rescue the ts mutants (Fig 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 from the putative mutants reestablished the ts phenotype when introduced into wild-type cells after being linearized. The two ts mutants isolated satisfied all of these criteria, establishing that they contained mutations associated specifically with the chromosomal dna2⁺ gene. These two ts mutants were named dna2-C1 and dna2-C2 (Fig 1A). The mutations were C-G to 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 Pro₉₅₆ and Leu₁₀₇₉ are conserved from budding yeast to human, and Pro₉₅₆ is located in the nucleotide-binding motif (Fig 1A). At the permissive temperature, cells carrying the dna2-C1 allele showed a slight growth defect, whereas those with the dna2-C2 allele showed no differences in growth, compared to wild-type cells (not shown). Following a shift to the nonpermissive temperature (37° for 6–8 hr), dna2-C2 cells arrested as elongated cells with a single nucleus (Fig 2C) that doubled their DNA content (measured by FACScan analyses, not shown). Cells carrying dna2-C1, subjected to the same analysis, yielded identical results (not shown). These findings are strikingly similar to those obtained with dna2::ura4⁺ disrupted spores (Fig 2A) and indicate that bulk chromosomal DNA replication occurs in the absence of a functional dna2⁺ product. These results are in accordance with those obtained for S. cerevisiae DNA2 (FIORENTINO and CRABTREE 1997 ), suggesting that the in vivo function of Dna2 is conserved in the two yeasts. We concluded that the dna2⁺ gene of S. pombe is not required for the initiation and elongation of replication forks, essential steps to replicate the chromosomal DNA en masse, regardless of modes of growth.

View larger version (49K): In this window In a new window Download PPT slide   Figure 3. Cells carrying a dna2-C2 mutant allele are sensitive to alkylating agents and a DNA replication inhibitor. Cells with either wild-type (wt) or mutant (dna2-C2, designated by C2) alleles of dna2+ were grown on minimal medium containing the indicated concentration of drugs at the permissive temperature (28°). The number of wild-type or mutant cells was first determined, and then serially diluted samples (104, 103, 102, and 10 cells) were spotted and grown for 4 days on plates containing the indicated concentrations of drugs, methyl methanesulfonate (MMS) or hydroxyurea (HU).

View larger version (87K): In this window In a new window Download PPT slide   Figure 4. Loss of dna2+ function triggers checkpoint control and results in chromosome breakage. (A) The control strain (hus1-14) or the double mutant HK12 strain containing hus1-14 dna2-C2 was grown at 25° and shifted to 37° for 8 hr. The cells were then stained with DAPI and examined under a microscope. Note that cells on the right were enlarged to observe the "cut" phenotypes (arrows) more closely. The double mutant cells contained heavily fragmented DNAs, which appear as speckled spots or unevenly distributed DNA. (B) Wild-type, HK10 (dna2-C2), and HK14 (cdc17-K42) cells grown in EMM supplemented with leucine and uracil at 25° were shifted to 37°. The cells were further incubated for 0 (lanes 1, 5, and 9), 2 (lanes 2, 6, and 10), 4 (lanes 3, 7, and 11), and 8 (lanes 4, 8, and 12) hr at 37°. Agarose plugs were prepared from these cells and the chromosomes were resolved by pulsed-field gel electrophoresis (PFGE) as described (GOULD et al. 1998 ). Each plug contained 108 cells and PFGE was carried out in 0.6% agarose in a CHEF-DR III apparatus (Bio-Rad, Richmond, CA) for 72 hr in 0.5x TAE buffer at 1.5 V/cm with a switch time of 30 min and 120° of included angle. In the case where the hydroxyurea arrest control was carried out (indicated as HU; lane 13), 12 mM (final concentration) was added to wild-type cultures grown at 25° and cells were further incubated for 4 hr at 30° prior to preparation of the agarose plug.

View larger version (28K): In this window In a new window Download PPT slide   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 dna2+, cdc27+, or cdc17+. Aliquots containing 104, 103, 102, and 10 transformed cells were spotted onto EMM plates containing leucine at the nonpermissive temperature of 34°. (B) The dna2-C2 mutant cells harboring vector (pREP3X) alone, pREP1-dna2+, pREP3X-cdc1+, or pREP41X-rad2+ under control of the nmt1 promoter were spotted (104, 103, 102, and 10 cells) and grown at 34° in the presence (+thi) or absence (-thi) of thiamine on EMM plates containing uracil. Note that the mutants containing dna2+ grow faster in the presence of thiamine (repressed condition) than in the absence of thiamine because overexpression of dna2+ caused growth retardation (FORMOSA and NITTIS 1999 ; PARENTEAU and WELLINGER 1999 ).

The temperature-sensitive mutation causes impaired resistance to distinct genotoxic agents:
Since inactivation of the dna2⁺ gene product did not affect DNA synthesis, we investigated the effects of genotoxic agents on HK10 dna2-C2 mutant strain (Table 1) in an effort to gain insight into the role(s) played by the dna2⁺ gene in DNA transactions other than replication. The experiments were done at the semipermissive temperature of 28°, which does not affect the growth of the dna2-C2 mutant (Fig 3). The dna2-C2 mutant was sensitive to methylmethane sulfonate (MMS) and slightly sensitive to 10 mM hydroxyurea (HU) but not to UV (doses ranging from 0 to 400 J/m²) compared to wild type (Fig 3). In view of its remarkable sensitivity to MMS, an alkylating agent, dna2⁺ is likely to play an important role in DNA repair, although the mechanism by which this occurs is unclear. The HK11 strain containing the dna2-C1 allele (Table 1) responded similarly to the various genotoxic agents tested above, like the dna2-C2 mutant (not shown).

The absence of dna2⁺ function triggers the replication checkpoint:
To test whether dna2⁺ is involved in DNA replication, we constructed a strain containing both dna2-C2 and hus1-14 mutations. The hus1⁺ gene plays a role in the DNA replication checkpoint: hus1-14 mutant cells with unreplicated DNA or damaged DNA fail to arrest at G2 and proceed into mitosis with fatal consequences (ENOCH et al. 1992 ). Thus, if dna2⁺ is required for DNA replication, the introduction of the checkpoint mutation, hus1-14, into cells containing the dna2-C2 mutation should allow the double mutant cells to enter mitosis, leading to catastrophic events at the nonpermissive temperature. Indeed, a significant percentage (>11%) of the hus1-14 dna2-C2 double mutant cells (HK12, Table 1) underwent aberrant mitosis when shifted to the nonpermissive temperature (37°), producing anucleated cells or progeny cells in which DNA was distributed unevenly (Fig 4A, right). In contrast, such aberrant mitosis was not detected in control cells containing the hus1-14 mutation alone (Fig 4A, left) or a dna2-C2 single mutant, which arrested as shown in Fig 2. The same catastrophic result was obtained when HK13 cells (Table 1) containing both dna2-C2 and rhp9::ura4⁺ were examined (not shown). The rhp9⁺ 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 37° most likely results in the incomplete replication of chromosomal DNA, which probably generates damaged DNA structures recognized by the DNA damage checkpoint. This suggests that nicks or ssDNA regions are present in the newly replicated DNA in the absence of dna2⁺. Therefore, we conclude that the dna2⁺ 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 incomplete chromosome replication:
The results described above indicate that the absence of dna2⁺ 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 Fig 4B, chromosomes from wild-type cells entered the gel and were separated into three chromosomes irrespective of the incubation temperature (Fig 4B, lanes 1–4). However, chromosomes isolated from dna2-C2 mutant cells (HK10) that were incubated >4 hr at the nonpermissive temperature failed to yield separated chromosomes (Fig 4B, lanes 7 and 8). Interestingly, the low molecular weight smear of DNA observed in the dna2-C2 mutant was similar to that found in cdc17-K42 cells (HK14) whose wild-type protein, DNA ligase I, functions in the maturation of Okazaki fragments (JOHNSTON et al. 1986 ; WAGA et al. 1994 ). The fragmented DNA in both cases accumulated at the bottom of the gel (Fig 4B). In addition, similarly fragmented DNA was also observed in cdc24 mutants whose growth defect was suppressed by dna2⁺ (GOULD et al. 1998 ). The smear of DNA may result from a qualitative defect of DNA replication such as pausing of replication forks exposing frail single-stranded DNA or generation of premature Okazaki fragments that have nicks or an unprocessed flap structure. Thus, the resulting DNA becomes prone to breakage by nuclease attack or fragile during experimental manipulation. It is also worthwhile to mention that the S. cerevisiae dna2-1 mutant synthesized only low molecular weight DNA at the nonpermissive 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 described above, indicate that the dna2-C2 mutation has a defect in DNA replication despite its ability to synthesize bulk DNA.

Isolation of multicopy suppressors for the dna2-C2 ts mutant:
An S. pombe genomic DNA library in the pUR19 vector was screened for genes that could rescue the temperature sensitivity of the dna2-C2 mutant using the procedures described in MATERIALS AND METHODS. The screening of 6.2 x 10⁵ HK10 cells transformed with the genomic library yielded 47 independent transformants that grew at the restrictive temperature. Among these candidate suppressors, 42 clones (~90%) contained wild-type dna2⁺ as expected, and five clones contained potential extragenic suppressors. Three clones had the complete sequence of the cdc17⁺ gene encoding DNA ligase I (JOHNSTON et al. 1986 ) in common. Two other clones contained both the complete sequence of the putative S. pombe fas gene encoding the folic acid synthesis protein (VOLPE et al. 1992 ) and the 5' two-thirds of the cdc27⁺ gene, a 54-kD subunit of the pol complex (MACNEILL et al. 1996 ). The latter two clones were analyzed further to confirm which gene was responsible for suppression. We found that cdc27⁺ alone was sufficient and necessary for the suppression of dna2-C2 mutation (not shown). The growth of the dna2-C2 mutant containing either cdc17⁺ or cdc27⁺ (the 5' two-thirds partial sequence) as a multicopy suppressor is shown in Fig 5A. In addition, the dna2-C1 mutation was also suppressed equally well by either cdc17⁺ or cdc27⁺ (not shown), suggesting that the defects associated with dna2-C1 and dna2-C2 are similar.

Since cdc27⁺ and cdc17⁺ genes are required for the elongation and maturation, respectively, of Okazaki fragments (ISHIMI et al. 1988 ; GOULIAN et al. 1990 ; WAGA and STILLMAN 1994 ; WAGA et al. 1994 ) and can suppress the defect observed with the two dna2 ts mutations, it is most likely that dna2⁺ functions in the Okazaki fragment metabolism. For this reason, we investigated whether other genes involved in Okazaki fragment metabolism also suppressed the temperature sensitivity of the dna2-C2 mutation. We constructed plasmids that contained pol3⁺, cdc1⁺, or cdm1⁺ (pol subunits) or rad2⁺ (an S. pombe homolog of RAD27) under the control of the nmt1 promoter (FORSBURG 1993 ; MAUNDRELL 1993 ). The wild-type dna2⁺ rescued the temperature-sensitive lethality regardless of the presence or absence of thiamine (Fig 5B). This is due to the high basal level activity of nmt1 promoter in pREP1 (MAUNDRELL 1993 ), which is sufficient to complement the dna2-C2 ts mutation. The dna2-C2 mutant cells did not grow at all when the expression of cdc1⁺ or rad2⁺ was not induced (Fig 5B). The induction of cdc1⁺ in the absence of thiamine allowed the dna2-C2 ts mutants to grow efficiently at the restrictive temperature (Fig 5B). In contrast, induction of the other pol subunits suppressed the growth defect of dna2-C2 weakly (cdm1⁺, encoding the 22-kD subunit of pol ) or not at all (pol3⁺, encoding the catalytic subunit of pol ; not shown). The rad2⁺ gene rescued the dna2-C2 defect when its expression was induced in the absence of thiamine (Fig 5B). These results demonstrate that the genes involved in the Okazaki fragment elongation or maturation genetically interact with dna2⁺.

The dna2-C2 mutation is synthetically lethal with cdc17-K42, rad2::ura4⁺, and cdc24-M38:
The data presented above support a role for dna2⁺ in the metabolism of Okazaki fragments. To further strengthen this conclusion, we examined whether the defect of dna2-C2 can be exaggerated when combined with mutant alleles of genes such as cdc17-K42 (DNA ligase I) and rad2::ura4⁺ (MURRAY et al. 1994 ). Tetrads obtained from the cross of dna2-C2 mutant cells (HK10) with either the cdc17-K42 (HK14) or rad2::ura4⁺ (HK15) mutant were dissected, and spores were grown at the permissive temperature of 25° (Table 2). In each case, a high proportion of dead spores was observed (Table 2; 29 and 21% inviable spores from 21 tetrads of HK10 x HK14 and 14 tetrads of HK10 x HK15 crosses, respectively). The percentage of dead spores from the two crosses was close to the expected percentage of dead spores (25%), assuming the combination of the two unlinked mutations is lethal. The dna2⁺ gene is on chromosome 2 and thus not linked to cdc17⁺, cdc24⁺, and rad2⁺, which are on chromosome 1. In test crosses between dna2-C2 and cdc17-K42 or between dna2-C2 and rad2::ura4⁺, no double mutants were detected among the growing spores, confirming that dna2-C2 is synthetically lethal when combined with cdc17-K42 or rad2::ura4⁺ (Table 2).

We also examined the genetic interaction between dna2⁺ and cdc24⁺, a novel replication gene of fission yeast essential for chromosome integrity (GOULD et al. 1998 ), to determine whether these two genes are synthetically lethal. For this purpose, we crossed dna2-C2 (HK10) and cdc24-M38 (HK16) mutant strains and analyzed the resulting tetrads. Among 15 tetrads dissected, 25% of the total spores obtained were inviable at 25° (Table 2) and no double mutant was found, suggesting a synthetic lethal interaction between the two genes. Fifteen tetrads from crossing of dna2-C2 with cdc27-P11 (MACNEILL et al. 1996 ) were also analyzed. Unlike the others above, dna2-C2 cdc27-P11 double mutants were recovered at the expected one-in-four frequency and viable at both 25° (permissive temperature) and 30° (semipermissive temperature). The failure to observe a synthetic lethal interaction between the two mutant alleles may be due to allele specificity. If, for example, a specific physical interaction is important for viability, only those mutant alleles that do not allow the physical interaction would cause the double mutants to be inviable. The synthetic lethality of dna2-C2 when combined with mutant alleles of several genes known to function in elongation or maturation of Okazaki fragments suggests that dna2⁺ is involved in DNA replication, especially at the stage of Okazaki fragment metabolism.

S. pombe Dna2 interacts with Cdc24, Cdc1, and Rad2 in the yeast two-hybrid system:
Since we confirmed the genetic interactions of dna2⁺ with cdc24⁺, rad2⁺, cdc27⁺, cdc1⁺, and cdc17⁺, we decided to investigate the physical interactions between dna2⁺ and those genes using the S. cerevisiae two-hybrid assay. We constructed a bait plasmid (pGBT9-dna2⁺) containing dna2⁺ fused to the GAL4 DNA-binding domain (BD) in pGBT9. The cdc24⁺, rad2⁺, cdc27⁺, cdc1⁺, and cdc17⁺ genes were fused to the GAL4 activation domain (AD) in pGAD424 or pACT2 to prepare prey plasmids (pGAD424-cdc24⁺, pACT2-rad2⁺, pGAD424-cdc27⁺, pGAD424-cdc1⁺, and pGAD424-cdc17⁺, respectively) as described in MATERIALS AND METHODS. The plasmid pGBT-dna2⁺ alone or in combination with pGAD424 or each plasmid expressing a prey protein did not activate transcription of reporter genes (Table 3 and not shown). When pGBT9-dna2⁺ (BD fusion) and pGAD424-cdc24⁺ (AD fusion) were cotransformed, a high level of ß-galactosidase activity was detected (Table 3). Consistent with this, cells cotransformed with pGBT9-dna2⁺ and pGAD424-cdc24⁺ turned blue within 1 hr when assayed for ß-galactosidase in filter assays (see MATERIALS AND METHODS), whereas control cells hardly turned blue even after 36 hr. This result indicates a strong interaction between S. pombe Dna2 and Cdc24. We observed that the reciprocal interaction using pGBT9-cdc24⁺ (BD fusion) and pGAD424-dna2⁺ (AD fusion) was weaker, but still significant (not shown). When pGBT9-dna2⁺ was cotransformed with either pGAD424-cdc1⁺ or pACT2-rad2⁺ (AD fusions), reduced levels of ß-galactosidase activity were detected (Table 3). Although these activities were relatively low, they were reproducibly ~10-fold higher than controls with either pGBT9-dna2⁺, pGAD424-cdc1⁺, or pACT2-rad2⁺ alone (Table 3 and not shown). This result suggests weak, but meaningful, interaction between the two proteins. In keeping with this, cells cotransformed with pGBT9-dna2⁺/pGAD424-cdc1⁺ and pGBT9-dna2⁺/pACT2-rad2⁺ turned blue within 8 hr in filter assays. In contrast, cells containing pGBT9-dna2⁺, pGAD424-cdc1⁺, or pACT2-rad2⁺ each alone did not develop blue color at >36 hr. The reciprocal combinations failed to lead to detectable ß-galactosidase activity (not shown), suggesting orientation-specific interactions in the two-hybrid assay. When pGBT9-dna2⁺ was cotransformed with either pGAD424-cdc27⁺ or pGAD424-cdc17⁺, the resulting transformants failed to show ß-galactosidase activity above background levels (Table 3). However, the cotransformed cells developed a pale blue color after prolonged incubation (>18 hr) in filter assays, which are ~10⁶ times more sensitive than the liquid assay (Table 3). These observations were highly reproducible and the control plasmid alone did not develop blue color even after >36 h of incubation (not shown).

To further confirm the interactions observed above, we examined the expression of the HIS3 reporter gene by growing cells in the presence of various concentrations of 3-aminotriazole (3-AT), which suppresses the leaky growth of false-positive cells (MANGUS et al. 1998 ). Cells containing each plasmid were grown to midlog phase and aliquots were spotted on SD plates containing increasing concentrations of 3-AT (Fig 6). The cells containing either vector (pGBT9, Fig 6) or bait alone (pGBT9-dna2⁺, not shown) failed to grow at high levels of 3-AT (Fig 6). However, cells containing pGBT9-dna2⁺ plus pGAD424-cdc24⁺, pACT2-rad2⁺, or pGAD424-cdc1⁺ grew efficiently in the presence of up to 30 mM 3-AT (Fig 6). At 20 mM 3-AT, the growth difference was indistinguishable in cells expressing cdc24⁺, rad2⁺, and cdc1⁺, whereas the growth of control cells containing vector alone was extremely poor. These results suggest that physical interactions exist between S. pombe Dna2 and Cdc24, Rad2, and Cdc1. We also investigated the interaction between the prey proteins, Cdc24, Rad2, Cdc27, and Cdc17, but did not detect any significant interaction among these proteins (not shown), suggesting that S. pombe Dna2 serves as a scaffold protein capable of interacting simultaneously with several proteins. The interactions observed in the yeast two-hybrid system are consistent with the abilities of cdc1⁺, cdc27⁺, cdc17⁺, and rad2⁺ to suppress the temperature lethality of dna2-C2. Two of these genes, cdc27⁺ and cdc17⁺, marginally interacted with dna2⁺; their interactions were detectable only in filter assays (Table 3). In conclusion, the specific genetic and two-hybrid interactions of dna2⁺ with genes known (DNA ligase, Fen-1, and pol ) or implicated (Cdc24) in Okazaki fragment metabolism place dna2⁺ as a protein that plays a critical role in Okazaki fragment elongation/maturation.

View larger version (115K): In this window In a new window Download PPT slide   Figure 6. The dna2+ interacts with cdc24+, rad2+, and cdc1+ in the yeast two-hybrid system. The budding yeast strain Y190 harboring pGBT9-dna2+ as bait was transformed with pGAD424 (vector), pGAD424-cdc24+, pGAD424-rad2+, and pGAD424-cdc1+ and the resulting transformants were examined for their ability to express the HIS3 reporter gene in the presence of increasing concentrations of 3-aminotriazole (3-AT). The cells (104, 103, 102, and 10) were spotted on synthetic minimal medium lacking L-tryptophan, L-leucine, and L-histidine and containing 3-AT at the concentrations indicated.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this article, we showed that S. pombe spores or vegetative cells with an inactivated dna2⁺ exhibited a distinct terminally arrested shape similar to that observed with the mutant cells defective in DNA replication genes such as cdc24⁺, cdc27⁺, or pcn1⁺ (WASEEM et al. 1992 ; MACNEILL et al. 1996 ; GOULD et al. 1998 ). They doubled their DNA content, however, suggesting that S. pombe Dna2 is not essential for the initiation of DNA replication or the progression of replication forks as suggested previously for S. cerevisiae Dna2 (FIORENTINO and CRABTREE 1997 ). Since the precise role of a protein in a multienzyme process such as DNA replication can be suggested by the proteins with which it interacts, we attempted to find those that can interact either genetically or physically with S. pombe Dna2. We concluded that S. pombe dna2⁺ plays a crucial role in the completion of DNA synthesis on the basis of the following results: (i) inactivation of S. pombe Dna2 triggered the replication checkpoint; (ii) loss of S. pombe dna2⁺ function caused qualitatively incomplete chromosome replication (chromosomes from the S. pombe dna2 mutant were heavily fragmented at the nonpermissive temperature); and (iii) S. pombe Dna2 interacted genetically with Rad2, DNA ligase I, and two subunits of pol (Cdc1 and Cdc27), all of which are involved directly in Okazaki fragment metabolism (WAGA and STILLMAN 1998 ). These findings in vivo are also consistent with the results from our studies in vitro showing that S. cerevisiae Dna2 is a single-stranded DNA-specific endonuclease that is well suited to completely remove primer RNA on Okazaki fragments (BAE et al. 1998 ; S.-H. BAE and Y.-S. SEO, unpublished results).

We observed an increased sensitivity of dna2-C2 mutants to MMS. Since MMS creates adducts and apurinic sites, which become single- and double-strand breaks that result from a failure to replicate past lesions containing 3-methyladenine (SCHWARTZ 1989 ), the dna2 mutants used in this study most likely lack the ability to remove damaged DNA prior to replicating the lesions, suggesting a role for Dna2 in DNA repair. The S. pombe mutant dna2 alleles that were isolated mapped inside the helicase domain (Fig 1), in keeping with the previous observation that MMS-sensitive alleles of S. cerevisiae DNA2 clustered in the helicase domain, and the site-directed motif I and motif II mutations caused MMS sensitivity (FORMOSA and NITTIS 1999 ). This result supports the notion that the helicase function is needed for some forms of DNA damage repair.

The ability of S. pombe dna2⁺ to interact with two subunits of pol , Rad2, and DNA ligase I raises the possibility that Dna2 exists in vivo within a multiprotein complex. On the basis of the results from yeast two-hybrid analyses (Table 3), however, interactions of S. pombe Dna2 with two subunits (Cdc1 and Cdc27) of pol may not be strong enough to allow formation of a stable complex between Dna2 and pol . Other interactions of S. pombe Dna2 with Rad2 and DNA ligase I would be necessary for Dna2 to be stably tethered to pol . Under these circumstances, the ability of Rad2 and DNA ligase I to complex directly with PCNA (LI et al. 1995 ; LEVIN et al. 1997 ; MONTECUCCO et al. 1998 ; GARY et al. 1999B ) could stabilize further the association of S. pombe Dna2 with pol , since PCNA itself is tightly coupled to pol while DNA synthesis occurs. This would account for the following discrepancy: we failed to observe any detectable complex formed between purified recombinant Rad27 and Dna2 of S. cerevisiae (not shown). In contrast, both Rad27 and S. cerevisiae Dna2 copurified on an immunoaffinity column, and they were reciprocally coimmunoprecipitated from crude extracts (BUDD and CAMPBELL 1997 ). In support of the hypothesis that Dna2 exists in a multiprotein complex, S. cerevisiae Dna2 in cell extracts eluted from a size-exclusion matrix column at ~700 kD, although this complex has not been analyzed yet (FORMOSA and NITTIS 1999 ).

Recent genetic studies on the mutant alleles of rad27 are also in accord with our hypothesis (GARY et al. 1999B ). The growth of mutant cells containing rad27-n (a nuclease-deficient allele of RAD27) was severely inhibited, whereas the growth of rad27 or rad27-p (defective in the PCNA-binding site) mutant cells was not (GARY et al. 1999A , GARY et al. 1999B ). The double mutant (rad27-n,p) was not inhibited. These observations were interpreted as follows. In rad27-n cells, the interaction of Rad27-n with PCNA allows the mutant protein to occupy its normal position within a multiprotein complex, which could hinder the action of secondary or redundant enzymes capable of processing the Okazaki fragment. In the case of rad27-n,p or rad27-p, the mutant proteins were not integrated within the multiprotein complex, hence allowing access of an alternative enzyme. These observations not only support the idea that processing of Okazaki fragments occurs in the context of a multiprotein assembly, but also predict the existence of a secondary processing enzyme that can operate in the absence of Fen-1 (Rad27). We believe that the enzyme is most likely Dna2 for the reasons described elsewhere (S.-H. BAE and Y.-S. SEO, unpublished results).

Dna2 may be regulated by proteins with which it interacts. Such a possibility is supported by significant differences in enzymatic activities noted between the recombinant S. cerevisiae Dna2 purified from insect cells and the one from S. cerevisiae cell extracts (BUDD and CAMPBELL 1995 ; BAE et al. 1998 ). Alternatively, Dna2 can alter enzymatic properties of proteins such as pol or Fen-1 via protein-protein interaction in order to coordinate the complicated multienzyme process of Okazaki fragment elongation and maturation. For example, the specific interaction between Dna2 and pol would lead to a change in the ability of pol to displace the 5'-end region of the preexisting Okazaki fragments. We showed that a flap structure generated by displacement DNA synthesis by pol was rapidly removed by S. cerevisiae Dna2 (S.-H. BAE and Y.-S. SEO, unpublished results). Considering that pol is capable of displacement DNA synthesis up to 274 bp, longer than the size (100–150 nucleotides) of Okazaki fragments (MOSSI et al. 1998 ), one possible consequence of Dna2-pol interaction would be the timely disassembly of the pol complex when no further displacement is required. At present, the roles played by Dna2 in the context of a multienzyme complex are highly conjectural and rigorous biochemical studies are needed to define any role of Dna2 in this regard. Recently, an additional role for Dna2 has been suggested by the observation that S. cerevisiae Dna2 interacts with POL1 and CTF4, which encode the DNA polymerase catalytic subunit and an associated protein, respectively (FORMOSA and NITTIS 1999 ). This suggests that Dna2 may also act in a process that involves pol in addition to the roles suggested above. Although pol -primase plays an essential role in Okazaki fragment initiation (TSURIMOTO et al. 1990 ; WAGA and STILLMAN 1994 ; WAGA et al. 1994 ), the precise link between Dna2, pol , and pol is not clearly understood.

There are a couple of differences noted in the DNA replication apparatus between fission yeast and budding yeast. For instance, S. pombe pol has one additional subunit, Cdm1, which has no structural counterpart in S. cerevisiae (MACNEILL et al. 1996 ; ZUO et al. 1997 ; GERIK et al. 1998 ). Another example is that S. cerevisiae lacks the structural equivalent of S. pombe cdc24⁺ (GOULD et al. 1998 ). The synthetic lethality of dna2-C2 cdc24-M38 and the two-hybrid interaction of S. pombe dna2⁺ with cdc24⁺ suggest that cdc24⁺ could affect the function of dna2⁺ and, like dna2⁺, it is most likely involved in Okazaki fragment metabolism. At the same time, these findings suggest that the mode of action of S. pombe Dna2 is likely to be different from that of S. cerevisiae Dna2. It is worthwhile to mention that the N-terminal 405 amino acids could be deleted without loss of any enzymatic activities of S. cerevisiae Dna2 (BAE et al. 1998 ), whereas deletion of >105 amino acids from its N terminus resulted in cell death (BUDD et al. 1995 ). These two observations suggest that the N-terminal region of S. cerevisiae Dna2 plays an essential regulatory role for Dna2 function, which may be required either to regulate the enzymatic activities of Dna2 or to serve as a site of protein-protein interaction. The N-terminal amino acid sequences are only weakly conserved among Dna2 homologs and do not contain motifs characteristic of any class of protein with known function. Therefore, the unique N-terminal amino acid sequence of each Dna2 protein may serve as a site of protein-protein interactions specific for its own regulatory protein. In keeping with this possibility, the cdc24⁺ gene specifically interacted with the N-terminal half of S. pombe dna2⁺ in the yeast two-hybrid assay (not shown). This may provide an explanation for the genetic and biochemical differences observed in the Dna2 proteins of fission yeast and budding yeast.

	ACKNOWLEDGMENTS

We thank Dr. Jerard Hurwitz for critical reading of the manuscript and comments on the manuscript. We also thank all the members of our laboratory for their valuable discussions. We are especially grateful to the members of laboratories of Drs. S. A. MacNeill, Y. Adachi, and P. A. Fantes in the University of Edinburgh for their kind help during the initial stage of this work. We thank the following people for providing strains, plasmids, and genomic DNA libraries: Dr. J. M. Murray and F. Z. Watts (University of Sussex, UK), Dr. H. Ohkura (University of Edinburgh, UK), Dr. J. Hurwitz (Sloan-Kettering Institute, USA), Dr. J. Rho (Seoul National University, Korea), and Dr. H. Yoo (Korea Research Institute of Bioscience and Biotechnology, Korea). We are greatly indebted to A. Sanderson (University of Edinburgh, UK) for FACScan analyses. This work was supported by a grant from the Creative Research Initiatives of the Korean Ministry of Science and Technology given to Y.-S.S.

Manuscript received November 17, 1999; Accepted for publication March 31, 2000.

	LITERATURE CITED

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