A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function.

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 delta temperature-sensitive growth defect. dna2-1 rad27/rth1 delta 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.     

Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway.     

Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis

Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase delta (Pol delta) with 5.-flap cutting by FEN1(RAD27) endonuclease. Excessive strand displacement is normally prevented by the 3.-exonuclease activity of Pol delta. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol delta) or PIF1 helicase genes can suppress lethality or growth defects of rad27Delta pol3-D520V mutants (defective for FEN1(RAD27) and the 3.-exonuclease of Pol delta) that produce long flaps and of dna2Delta mutants that are defective in cutting long flaps. On the contrary, pol32Delta or pif1Delta caused lethality of rad27Delta exo1Delta double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1(RAD27) and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol delta is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1(RAD27) during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol delta to FEN1(RAD27). Finally, RNA-DNA hybrids are more readily displaced by Pol delta than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation.     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.     

Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta

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 pif1delta dna2delta strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol delta, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32delta strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol delta and/or by an as yet unidentified helicase. Thus, suppression of dna2delta 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 pif1Delta mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5′ to 3′ DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5′ to 3′ exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5′ to 3′ helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.Key words: yeast, RAD27, RAD9, RAD53, Okazaki fragment processing, DNA replication, exo1     

Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae

The SRS2 gene of Saccharomyces cerevisiae encodes a DNA helicase that is active in the postreplication repair pathway and homologous recombination. srs2 mutations are lethal in a rad54Delta background and cause poor growth or lethality in rdh54Delta, rad50Delta, mre11Delta, xrs2Delta, rad27Delta, sgs1Delta, and top3Delta backgrounds. Some of these genotypes are known to be defective in double-strand break repair. Many of these lethalities or poor growth can be suppressed by mutations in other genes in the DSB repair pathway, namely rad51, rad52, rad55, and rad57, suggesting that inhibition of recombination at a prior step prevents formation of a lethal intermediate. Lethality of the srs2Delta rad54Delta and srs2Delta rdh54Delta double mutants can also be rescued by mutations in the DNA damage checkpoint functions RAD9, RAD17, RAD24, and MEC3, indicating that the srs2 rad54 and srs2 rdh54 mutant combinations lead to an intermediate that is sensed by these checkpoint functions. When the checkpoints are intact the cells never reverse from the arrest, but loss of the checkpoints releases the arrest. However, cells do not achieve wild-type growth rates, suggesting that unrepaired damage is still present and may lead to chromosome loss.     

Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Delta double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Delta alone. However, surprisingly, the dna2-2 sgs1Delta lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Delta lethality is only partially suppressed by deletion of rad51Delta. We propose that the replication-associated defects that we document in the rDNA are characteristic 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.     

Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging.

The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.     

Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth

Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomyces cerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol alpha. DNA2 alleles were isolated that cause either temperature sensitivity, sensitivity to alkylation damage, or both. The alkylation-sensitive alleles clustered in the helicase domain, including changes in residues required for helicase activity in related proteins. Additional mutations known or expected to destroy the ATPase and helicase activities of Dna2 were constructed and found to support growth on some media but to cause alkylation sensitivity. Only damage-sensitive alleles were lethal in combination with a ctf4 deletion. Full activity of the Dna2 helicase function is therefore not needed for viability, but is required for repairing damage and for tolerating loss of Ctf4. Arrest of dna2 mutants was RAD9 dependent, but deleting this checkpoint resulted in either no effect or suppression of defects, including the synthetic lethality with ctf4. Dna2 therefore appears to act in repair or lagging strand synthesis together with Pol alpha and Ctf4, in a role that is optimal with, but does not require, full helicase activity.     

Single strand annealing and ATP-independent strand exchange activities of yeast and human DNA2: possible role in Okazaki fragment maturation

The Dna2 protein is a multifunctional enzyme with 5'-3' DNA helicase, DNA-dependent ATPase, 3' exo/endonuclease, and 5' exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented.     

The short life span of Saccharomyces cerevisiae sgs1 and srs2 mutants is a composite of normal aging processes and mitotic arrest due to defective recombination

Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.     

Helicase and nuclease activities of hyperthermophile Pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments at 5'-end

Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya. Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophile Pyrococcus horikoshii (Dna2Pho). Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae. Dna2Pho has single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase activity (5' to 3' direction) requiring ATP, and nuclease activity, which prefers free 5'-ends of ssDNA as substrate. These activities depend on MgCl(2) concentrations. Dna2Pho requires a higher concentration of MgCl(2) for the nuclease than helicase activity. Both the helicase and nuclease activities of Dna2Pho were inhibited by substrates with RNA segments at the 5'-end of flap DNA, whereas the nuclease activity of Dna2 from S. cerevisiae was reported to be stimulated by RNA segments in the 5'-tail (Bae, S.-H., and Seo, Y. S. (2000) J. Biol. Chem. 38022-38031).     

Cross talk between the nuclease and helicase activities of Dna2: role of an essential iron-sulfur cluster domain

Dna2 nuclease/helicase is a multitasking protein involved in DNA replication and recombinational repair, and it is important for preservation of genomic stability. Yeast Dna2 protein contains a conserved putative Fe-S (iron-sulfur) cluster signature motif spanning the nuclease active site. We show that this motif is indeed an Fe-S cluster domain. Mutation of cysteines involved in metal coordination greatly reduces not just the nuclease activity but also the ATPase activity of Dna2, suggesting that the nuclease and helicase activities are coupled. The affinity for DNA is not significantly reduced, but binding mode in the C to A mutants is altered. Remarkably, a point mutation (P504S), proximal to the Fe-S cluster domain, which renders cells temperature sensitive, closely mimics the global defects of the Fe-S cluster mutation itself. This points to an important role of this conserved proline residue in stabilizing the Fe-S cluster. The C to A mutants are deficient in DNA replication and repair in vivo, and, strikingly, the degree to which they are defective correlates directly with degree of loss of enzymatic activity. Taken together with previous results showing that mutations in the ATP domain affect nuclease function, our results provide a new mechanistic paradigm for coupling between nuclease and helicase modules fused in the same polypeptide.     

Characterization of Saccharomyces cerevisiae dna2 Mutants Suggests a Role for the Helicase Late in S Phase

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like “lipid kinase” domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.     

Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3′-GT-overhangs that extend beyond the complementary 5′-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G₂ phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5′-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5′-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.     

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair.

Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of the RAD2 gene in S. cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains. In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease. Expression of the EXO1 gene is UV inducible. Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants. Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA. The exo1 msh2 , exo1 rad51 , rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants. This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER. Further, it suggests that the EXO1 , RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.     

Nej1 interacts with Sae2 at DNA double-stranded breaks to inhibit DNA resection

The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5′-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5′-resection mediated by the nuclease/helicase complex Dna2–Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11–Rad50–Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs.