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.     

The pattern of sensitivity of yeast dna2 mutants to DNA damaging agents suggests a role in DSB and postreplication repair pathways

The Saccharomyces cerevisiae DNA2 gene encodes a DNA-stimulated ATPase and DNA helicase/nuclease essential for DNA replication. In characterizing dna2 mutants, we have found that Dna2p also participates in DNA repair or in damage avoidance mechanisms. dna2 mutants are sensitive to X rays, although they are less sensitive than rad52 mutants. The X-ray sensitivity of dna2 mutants is suppressed by overexpression of a 5' to 3' exonuclease, the yeast FEN-1 structure-specific nuclease, encoded by the RAD27 gene, which also suppresses the growth defect of dna2-ts mutants. SGS1 encodes a helicase with similar properties to Dna2 protein. Although sgs1Delta mutants are resistant to X rays, dna2-2 sgs1Delta double mutants are more sensitive to X rays than the dna2-2 mutant. Temperature sensitive dna2 mutants are only slightly sensitive to UV light, show normal levels of spontaneous and UV induced mutagenesis, and have only a 2.5-fold elevated level of dinucleotide tract instability compared to wildtype. However, dna2Delta strains kept alive by overproduction of RAD27 are highly sensitive to UV light. These phenotypes, in addition to the epistasis analysis reported, allow us to propose that Dna2 is involved in postreplication and DSB repair pathways.     

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     

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.     

Genetic and functional interactions between Mus81–Mms4 and Rad27

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81–Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81–Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81–Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81–Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81–Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein–protein interaction between the two proteins. Our in vitro data indicate that Mus81–Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81–Mms4 in context of DNA replication.     

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.     

Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism

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 delta (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.     

Genetics of lagging strand DNA synthesis and maturation in fission yeast: suppression analysis links the Dna2-Cdc24 complex to DNA polymerase delta

The Cdc24 protein is essential for the completion of chromosomal DNA replication in fission yeast. Although its precise role in this process is unclear, Cdc24 forms a complex with Dna2, a conserved endonuclease–helicase implicated in the removal of the RNA–DNA primer during Okazaki fragment processing. To gain further insights into Cdc24–Dna2 function, we screened for chromosomal suppressors of the temperature-sensitive cdc24-M38 allele and mapped the suppressing mutations into six complementation groups. Two of these mutations defined genes encoding the Pol3 and Cdc27 subunits of DNA polymerase δ. Sequence analysis revealed that all the suppressing mutations in Cdc27 resulted in truncation of the protein and loss of sequences that included the conserved C-terminal PCNA binding motif, previously shown to play an important role in maximizing enzyme processivity in vitro. Deletion of this motif is shown to be sufficient for suppression of both cdc24-M38 and dna2-C2, a temperature-sensitive allele of dna2⁺, suggesting that disruption of the interaction between Cdc27 and PCNA renders the activity of the Cdc24–Dna2 complex dispensable.     

Identification of the Xenopus laevis homolog of Saccharomyces cerevisiae DNA2 and its role in DNA replication

The DNA2 gene of Saccharomyces cerevisiae is essential for growth and appears to be required for a late stage of chromosomal DNA replication. S. cerevisiae Dna2p (ScDna2p) is a DNA helicase and also a nuclease. We have cloned and sequenced the homologous gene from Xenopus (Xenopus Dna2). Xenopus Dna2p (XDna2p) is 32% identical to ScDna2p, and the similarity extends over the entire length, including but not limited to the five conserved helicase motifs. XDna2p is even more closely related (60% identical) to a partial human cDNA. The Xenopus Dna2 (XDna2) gene was able to complement an S. cerevisiae dna2-1 mutant strain for growth at the nonpermissive temperature, suggesting that XDna2p is a functional as well as a structural homolog of the yeast protein. Recombinant XDna2p was expressed in insect cells and purified. Like the ScDna2p purified from yeast, it is a single-stranded DNA endonuclease and a DNA-dependent ATPase, suggesting that both of these activities are part of the essential function of Dna2p. However, unlike ScDna2p from yeast, recombinant XDna2p showed no DNA helicase activity. When XDna2 was immunodepleted from interphase egg extracts, chromosomal DNA replication was almost completely inhibited. From the size of the residually synthesized DNA from the XDna2-depleted egg extracts, it seems that initiation of DNA replication may be impaired. This interpretation is also supported by the normal DNA replication of M13 single-stranded DNA in the XDna2-depleted egg extracts.     

Human Dna2 Is a Nuclear and Mitochondrial DNA Maintenance Protein

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance.DNA damage arises from errors in the replication process, as well as a myriad of intrinsic and extrinsic DNA-damaging agents that continually assault cells. Failure to efficiently repair DNA lesions leads to accumulation of mutations that contribute to numerous pathologies, including carcinogenesis. In addition to genomic DNA, mitochondrial DNA (mtDNA) is subject to damage that requires repair to maintain integrity. For these reasons, it is not surprising that DNA replication and repair proteins display significant plasticity that allows participation in several divergent replication and repair processes. In addition, numerous mechanisms, including alternative splicing, posttranslational modifications, or utilization of alternative translation initiation start sites, allow DNA replication and repair proteins such as Pif1, DNA ligase III, and APE1 to localize to the nucleus and the mitochondrion and participate in DNA replication and/or repair (9, 17, 25), thus ensuring genomic DNA and mtDNA integrity.Dna2 is an evolutionarily conserved helicase/nuclease enzyme. Originally discovered in Saccharomyces cerevisiae, Dna2 orthologs are found throughout the animal kingdom, including humans (5, 22, 28). Early studies demonstrated that Dna2 functions in concert with Flap endonuclease 1 (FEN1) to remove long DNA flaps that form upon lagging-strand DNA replication (6). However, in contrast to FEN1, Dna2 is an essential gene in yeast, suggesting that other proteins, including FEN1, cannot compensate for its loss in DNA replication or that it possesses functions beyond its role in Okazaki fragment processing. In agreement with this, genetic and biochemical studies have implicated Dna2 in DNA double-strand break (DSB) repair, telomere regulation, and mitochondrial function (8, 10, 15, 26, 38, 44, 45).Analysis of Dna2 in yeast revealed that it undergoes dynamic cell cycle localization. Dna2 localizes to telomeres during G₁, relocalizes throughout the genome in S phase, and moves back to the telomere during late S/G₂, where it participates in telomere replication and telomerase-dependent telomere elongation (10). Dna2 also leaves the telomere following treatment with bleomycin and localizes to sites of DNA DSBs (10). In addition, dna2 mutants are sensitive to DNA damage induced by gamma radiation and methanesulfonic acid methyl ester (7, 15). These phenotypes may be explained by recent work demonstrating that Dna2 plays an important role in 5′-end resection following DSBs. Indeed, upon induction of DSBs and initiation of 5′-end resection by the Mre11-Rad50-Xrs2 complex, Dna2 and Sgs1 cooperate to further degrade the 5′ end, creating long 3′ strands essential for homologous recombination (26, 45). Finally, while dna2Δ mutations are lethal in budding yeast, the dna2Δ pif1-m2 (nuclear PIF1) double mutations rescue dna2Δ lethality but produce a petite phenotype, suggesting that Dna2 is also involved in mtDNA maintenance (8).Recently, the human ortholog of Dna2 was cloned and characterized (23, 29). Biochemical analysis revealed that, similar to its yeast counterpart, the human Dna2 (hDna2) protein possesses nuclease, ATPase, and limited helicase activities (23, 29), suggesting that it carries out analogous functions in yeast and mammalian cells. However, hDna2''s putative role in genomic DNA repair and replication was called into question by a recent study suggesting that hDna2 is absent from the nucleus and found exclusively within the mitochondria, where it participates in mtDNA repair (44). Further in vitro biochemical studies suggested that hDna2 also participates in mtDNA replication (44). Here, we confirm that hDna2 localizes to the mitochondria and demonstrate that hDna2 participates in mtDNA replication and repair. However, our studies go further by uncovering a nuclear form of hDna2 that plays an important role in genomic stability. Indeed, we demonstrate that depletion of hDna2 leads to the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that hDna2, like its yeast counterpart, is essential to maintain nuclear DNA stability.     

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.     

The trans-autostimulatory activity of Rad27 suppresses dna2 defects in Okazaki fragment processing

Dna2 and Rad27 (yeast Fen1) are the two endonucleases critical for Okazaki fragment processing during lagging strand DNA synthesis that have been shown to interact genetically and physically. In this study, we addressed the functional consequences of these interactions by examining whether purified Rad27 of Saccharomyces cerevisiae affects the enzymatic activity of Dna2 and vice versa. For this purpose, we constructed Rad27DA (catalytically defective enzyme with an Asp to Ala substitution at amino acid 179) and found that it significantly stimulated the endonuclease activity of wild type Dna2, but failed to do so with Dna2Δ405N that lacks the N-terminal 405 amino acids. This was an unexpected finding because dna2Δ405N cells were still partially suppressed by overexpression of rad27DA in vivo. Further analyses revealed that Rad27 is a trans-autostimulatory enzyme, providing an explanation why overexpression of Rad27, regardless of its catalytic activity, suppressed dna2 mutants as long as an endogenous wild type Rad27 is available. We found that the C-terminal 16-amino acid fragment of Rad27, a highly polybasic region due to the presence of multiple positively charged lysine and arginine residues, was sufficient and necessary for the stimulation of both Rad27 and Dna2. Our findings provide further insight into how Dna2 and Rad27 jointly affect the processing of Okazaki fragments in eukaryotes.     

Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

The Schizosaccharomyces pombe pfh1⁺ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1⁺ gene suppress a temperature-sensitive allele of cdc24⁺, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1⁺ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1⁺ and dna2⁺ (or cdc24⁺) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol δ-mediated lagging strand displacement DNA synthesis.     

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.     

Enzymatic properties of the Caenorhabditis elegans Dna2 endonuclease/helicase and a species-specific interaction between RPA and Dna2

In both budding and fission yeasts, a null mutation of the DNA2 gene is lethal. In contrast, a null mutation of Caenorhabditis elegans dna2⁺ causes a delayed lethality, allowing survival of some mutant C.elegans adults to F2 generation. In order to understand reasons for this difference in requirement of Dna2 between these organisms, we examined the enzymatic properties of the recombinant C.elegans Dna2 (CeDna2) and its interaction with replication-protein A (RPA) from various sources. Like budding yeast Dna2, CeDna2 possesses DNA-dependent ATPase, helicase and endonuclease activities. The specific activities of both ATPase and endonuclease activities of the CeDna2 were considerably higher than the yeast Dna2 (~10- and 20-fold, respectively). CeDna2 endonuclease efficiently degraded a short 5′ single-stranded DNA tail (<10 nt) that was hardly cleaved by ScDna2. Both endonuclease and helicase activities of CeDna2 were stimulated by CeRPA, but not by human or yeast RPA, demonstrating a species-specific interaction between Dna2 and RPA. These and other enzymatic properties of CeDna2 described in this paper may shed light on the observation that C.elegans is less stringently dependent on Dna2 for its viability than Saccharomyces cerevisiae. We propose that flaps generated by DNA polymerase δ-mediated displacement DNA synthesis are mostly short in C.elegans eukaryotes, and hence less dependent on Dna2 for viability.     

Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro

We have previously shown that replication- protein A (RPA), the heterotrimeric single-stranded DNA binding protein of eukaryotes, plays a role in Okazaki fragment processing by acting as a molecular switch between the two endonucleases, Dna2 and Fen1, to ensure the complete removal of primer RNAs in Saccharomyces cerevisiae. The stimulation of Dna2 endonuclease activity by RPA requires direct protein–protein interaction. In this report we have analyzed genetically and biochemically the interaction of Dna2 with RPA. RFA1, the gene encoding the large subunit of RPA, displayed allele-specific interactions with DNA2 that included synthetic lethality and intergenic complementation. In addition, we identified physical and functional interactions between these proteins and found that RPA binds Dna2 predominantly through its large subunit, Rpa1. Consistent with the mapping of synthetic lethal mutations, robust interaction localizes to the C-termini of these proteins. Moreover, the N-terminal domains of Dna2 and Rpa1 appear to be important for a functional interaction because the N-terminal domain of RPA1 was required to maximally stimulate Dna2 endonuclease activity. We propose that a bimodal interaction of Dna2 with Rpa1 is important for Dna2 function both in vivo and in vitro. The relevance of each interaction with respect to the function of the Dna2 endonuclease activity is discussed.     

Dna2 requirement for normal reproduction of Caenorhabditis elegans is temperature-dependent

To determine the function of Dna2 in a multicellular organism, the Caenorhabditis elegans Dna2 expression was probed and deletion mutant phenotypes were analyzed. Dna2 was localized to the nuclei of C. elegans oocytes and early embryos by immunostaining or green fluorescent protein-tagging. A homozygous dna2 deletion mutant showed a reduced brood size and embryonic lethality, and the phenotypes greatly depended on growth temperature and aggravated in the succeeding generation. The mutant embryos also showed delayed cell divisions, which together with temperature-dependence of the mutant phenotypes supported the well-conserved role of Dna2 in DNA replication.