The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1⌬ dna2⌬ strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol ␦, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32⌬ strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol ␦ and/or by an as yet unidentified helicase. Thus, suppression of dna2⌬ can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1⌬ mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.Yeast Pif1 is the founding member of the Pif1 subfamily of superfamily 1 DNA helicases (3). While other organisms, such as Caenorhabditis elegans and Homo sapiens, have only one identified Pif1 family member, in yeast, there is a second, closely related, protein, Rrm3p (3). In yeast, neither of these helicases is essential and mutants lacking both are viable and repair proficient. Both yeast proteins have 5Ј-to-3Ј DNA helicase activity (21,30,31). The region of similarity between Pif1 and Rrm3 is limited to the seven helicase motifs, which exhibit 40% identity and 60% similarity (3). This may indicate that the two helicases have structurally similar DNA substrates. Nevertheless, the two helicases differ in their biological functions, and these differences are likely mediated not only by the helicase domain but also by the divergent N termini, which are not required for helicase activity (4). To date, it has been impossible to determine which helicase, Rrm3 or Pif1, is the functional homolog of the single ortholog in other eukaryotes.One difference between Rrm3 and Pif1 is in their function at the rRNA gene. In rrm3 mutants, there is an increase in replisome pausing at the Fob1 protein-bound replication fork barrier (RFB) in the rRNA gene (22). The hypothesis is that Rrm3 is required to remove proteins that block the fork at that point, since Rrm3 is required for promoting fork movement at over 1,400 loci in the yeast genome, in addition to the RFB. In contrast to Rrm3, Pif1 seems to be required for pausing at the rRNA...

SUMMARY Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase ε, essential for leading strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replication forks stalled in HU. The bimodal Mrc1/Pol2 interaction may identify a novel step in regulating the S phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, which also interacts with the MCMs, may modulate coupling of polymerization and unwinding at the replication fork.

Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease. To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease. We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells. A mutation in the Walker A box in the Cterminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease. Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site. Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP. These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP. Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair. Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype.Yeast dna2-1 mutants were originally identified in a screen for mutants defective in DNA replication in vitro (1) and were then shown to be defective in DNA replication in vivo (1, 2). Since that time, additional dna2 mutants with similar phenotypes have been identified and characterized (3, 4). Fluorescence-activated cell sorting analysis shows that temperaturesensitive dna2 mutants can synthesize a full 2C DNA content at 37°C (3).1 The DNA synthesized is highly fragmented, however, indicating that, although there is extensive DNA synthesis, DNA replication is incomplete in some way (2). Strains with dna2 deletions are inviable, showing Dna2 performs an essential function during DNA replication (2-4). Recently, it has also been demonstrated that dna2 mutants are defective in repair of x-ray-, bleomycin-, and methylmethane sulfonateinduced DNA damage (4, 5).Dna2 is a 170-kDa protein with six motifs characteristic of DNA helicases in the C-terminal third of the protein. A schematic diagram of the protein is shown in Fig. 1. Genes homologous to DNA2 have been identified in Schizosaccharomyces pombe, Xenopus laevis, Caenorhabditis elegans, and humans (6 -8). Immunoaffinity-purified Dna2 has DNA-dependent ATPase activity, DNA helicase activity that requires a 5Ј nonhybridized tail adjacent to the duplex region unwound, and a potent endonuclease activity (2, 9 -11). The helicase and ...

We have recently described a new helicase, the Dna2 helicase, that is essential for yeast DNA replication. We now show that the yeast FEN-1 (yFEN-1) nuclease interacts genetically and biochemically with Dna2 helicase. FEN-1 is implicated in DNA replication and repair in yeast, and the mammalian homolog of yFEN-1 (DNase IV, FEN-1, or MF1) participates in Okazaki fragment maturation. Overproduction of yFEN-1, encoded by RAD27/RTH1, suppresses the temperature-sensitive growth of dna2-1 mutants. Overproduction of Dna2 suppresses the rad27/rth1⌬ temperature-sensitive growth defect. dna2-1 rad27/rth1⌬ double mutants are inviable, indicating that the mutations are synthetically lethal. The genetic interactions are likely due to direct physical interaction between the two proteins, since both epitope-tagged yFEN-1 and endogenous yFEN-1 coimmunopurify with tagged Dna2. The simplest interpretation of these data is that one of the roles of Dna2 helicase is associated with processing of Okazaki fragments.

A yeast gene has been identified by screening for DNA replication mutants using a permeabilized cell replication assay. The mutant is temperature sensitive for growth and shows a cell cycle phenotype typical of DNA replication mutants. RNA synthesis is normal in the mutant but DNA synthesis ceases upon shift to the nonpermissive temperature. The DNA2 gene was cloned by complementation of the dna2, gene phenotype. The gene is essential for viability. The gene encodes a 172-kDa protein with characteristic DNA helicase motifs. A hemagglutinin epitope-Dna2 fusion protein was prepared and purified by conventional and immunoaffinity chromatography. The purified protein is a DNAdependent ATPase and has 3' to 5' DNA helicase activity specific for forked substrates. A nuclease activity that endonucleolytically cleaves DNA molecules having a singlestranded 5' tail adjacent to a duplex region copurifies through all steps with the fusion protein.In the absence of eukaryotic in vitro replication systems dependent on chromosomal origins of replication, the genetic approach offered by Saccharomyces cerevisiae provides an alternative way to identify and analyze the cellular proteins involved in replication. Homologs of the cellular proteins and genes encoding all of the activities required for simian virus 40 leading-strand DNA synthesis have been identified in yeast: DNA polymerase a/primase, DNA polymerase 8 and its accessory proteins (RF-C and PCNA), and the three subunits of the single-stranded DNA binding protein RP-A (see ref. 1 for review). A third DNA polymerase, s, is also required for chromosomal synthesis in yeast, but its specificity for lagging-or leading-strand synthesis is not clear. A yeast origin recognition complex has also been described (2). However, of the major proteins involved in assembling and moving replication forks, a helicase activity has thus far eluded discovery in yeast. It is likely that additional components also remain to be identified. This situation has fostered efforts to identify additional replication genes through isolation of new yeast replication mutants.We have previously described a screen for DNA replication mutants that monitors DNA synthesis in detergentpermeabilized cells as an assay for DNA replication (3). DNA synthesis in the permeabilized cells is dependent on addition of ATP and all four deoxynucleoside triphosphates. It is thought to represent continuation of synthesis at replication forks active in vivo at the time of permeabilization, and indeed the permeabilized cells remain viable and grow when diluted into fresh growth medium. A collection of 400 temperaturesensitive mutants generated by mutagenesis with nitrosoguanidine and previously screened for a specific cell division cycle defect-i.e., arrest with a uniform morphology at the nonpermissive temperature-was used in our study (4). Reasoning that some DNA replication mutants might not arrest uniformly in the cell cycle and thus might have been missed in the cdc screen, we grew each strain at the permissive tem...

Although a number of eukaryotic DNA helicases have been identified biochemically and still more have been inferred from the amino acid sequences of the products of cloned genes, none of the cellular helicases or putative helicases has to date been implicated in eukaryotic chromosomal DNA replication. By the same token, numerous eukaryotic replication proteins have been identified, but none of these is a helicase. We have recently identified and characterized a temperature-sensitive yeast mutant, Here we show that the helicase domain is required in vivo and that a 3 to 5 DNA helicase activity specific for forked substrates is intrinsic to the Dna2p. The N terminus is also essential for DNA replication. Thus, the structure of this new helicase is different from all previously characterized replicative helicases, which is consistent with the complex organization of eukaryotic replication forks, where the activities of not one but three essential DNA polymerases must be coordinated.A DNA helicase is a central component of the architecture of prokaryotic DNA replication forks. Reconstitution of the basal apparatus for replication of the SV40 virus has established the requirement for a DNA helicase in eukaryotic DNA replication as well. However, SV40 DNA replication requires only the helicase associated with the viral large T antigen and no cellular helicase. Therefore, we have looked for a cellular replicative helicase using yeast genetic analysis.Recently, we characterized a gene, DNA2, which complements a temperature-sensitive yeast strain defective in the elongation stage of DNA replication (1, 2). The DNA2 gene is essential for viability and encodes a 1522-amino acid protein, the most prominent feature of which is the presence of the six conserved motifs characteristic of DNA helicases. These motifs are localized to the COOH third of the protein (amino acids . In order to demonstrate that the protein had helicase activity, the HA-Dna2 protein was purified 50,000-fold. The immunoaffinity-purified protein was shown to be associated with a DNA-dependent ATPase and a DNA helicase. Interestingly, the helicase is active only on a substrate with a forked structure, as is true of many prokaryotic and viral replicative helicases and appears to translocate in the 3Ј to 5Ј direction, the polarity of the leading strand at a replication fork (2).While these experiments suggest that Dna2p is a replicative helicase, they are preliminary in two ways. First, although mock purifications yield no ATPase or helicase activity, our biochemical approach could not rule out that the ATPase and helicase activities were copurifying with, rather than intrinsic to, the Dna2p. Second, because more than two-thirds of the protein sequence was not conserved in any known helicase and might therefore encode some novel replicative function, our previous results did not allow us to conclude that the essential role of Dna2p in replication was that of a helicase. An example of a DNA-dependent ATPase and helicase whose essential function may not requir...

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of doublestrand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with ⌬rrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.Replication fork stress has been implicated as a major cause of genome instability in bacteria and yeast. In Escherichia coli, replication forks initiated at the origins frequently stall because of mutations in replication proteins, template blocks, or pauses at natural replication terminator sites. A common intermediate in restoring replication forks after stalling is a double-strand break (DSB), 1 which is thought to lead to recombination, producing genomic instability.Evidence that replication forks pause in Saccharomyces cerevisiae is also convincing (1, 2). In the presence of the replication inhibitor HU, forks stall and give rise to single-stranded regions at the forks. In the absence of checkpoint function, the stalled forks are converted to regressed forks, a Holliday-like structure arising by branch migration and reannealing of nascent DNA strands (3). Primase mutants also show high levels of stalled and regressed forks (1). There is also a naturally occurring replication fork barrier (RFB) within the rDNA (ribosomal DNA) repeats, and the structure of forks paused at the RFB has been characterized (4 -6). Finally, recent evidence suggests that the yeast ATR homolog, Mec1, protects replication forks from collapsing and giving rise to DSBs in replication slow zones throughout the chromosome (7). Evidence is also accumulating that replication fork failure leads to recombinogenic structures that result in gross chromosomal rearrangements (7-10). Such events may lead to the well documented genomic instability observed in DNA replication and checkpoint mutants (8, 9). We have proposed that replication fork stallin...

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