Copyright 2000 by the Genetics Society of America Genetic Analyses of Schizosaccharomyces pombe dna2؉ Reveal That Dna2 Plays an Essential Role in Okazaki Fragment Metabolism Ho-Young Kang,*,² Eunjoo Choi,* Sung-Ho Bae,* Kyoung-Hwa Lee,* Byung-Soo Gim,* Hee-Dai Kim,* Chankyu Park,² Stuart A. MacNeill³ 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, ²Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusung-Ku, Taejon, 305-701, Korea and ³Institute 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 de®ne 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- ®ed 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 pro- liferating 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 ®lling 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-speci®c 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 ¯ap 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, ®sh, 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 speci®c 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).