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1.  Human MUS81-EME2 can cleave a variety of DNA structures including intact Holliday junction and nicked duplex 
Nucleic Acids Research  2014;42(9):5846-5862.
MUS81 shares a high-degree homology with the catalytic XPF subunit of the XPF–ERCC1 endonuclease complex. It is catalytically active only when complexed with the regulatory subunits Mms4 or Eme1 in budding and fission yeasts, respectively, and EME1 or EME2 in humans. Although Mus81 complexes are implicated in the resolution of recombination intermediates in vivo, recombinant yeast Mus81-Mms4 and human MUS81-EME1 isolated from Escherichia coli fail to cleave intact Holliday junctions (HJs) in vitro. In this study, we show that human recombinant MUS81-EME2 isolated from E. coli cleaves HJs relatively efficiently, compared to MUS81-EME1. Furthermore, MUS81-EME2 catalyzed cleavage of nicked and gapped duplex deoxyribonucleic acids (DNAs), generating double-strand breaks. The presence of a 5′ phosphate terminus at nicks and gaps rendered DNA significantly less susceptible to the cleavage by MUS81-EME2 than its absence, raising the possibility that this activity could play a role in channeling damaged DNA duplexes that are not readily repaired into the recombinational repair pathways. Significant differences in substrate specificity observed with unmodified forms of MUS81-EME1 and MUS81-EME2 suggest that they play related but non-overlapping roles in DNA transactions.
doi:10.1093/nar/gku237
PMCID: PMC4027171  PMID: 24692662
2.  Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase 
Nucleic Acids Research  2013;41(6):3576-3587.
DNA repair helicases function in the cell to separate DNA duplexes or remodel nucleoprotein complexes. These functions are influenced by sensing and signaling; the cellular pool of a DNA helicase may contain subpopulations of enzymes carrying different post-translational modifications and performing distinct biochemical functions. Here, we report a novel experimental strategy, single-molecule sorting, which overcomes difficulties associated with comprehensive analysis of heterologously modified pool of proteins. This methodology was applied to visualize human DNA helicase F-box–containing DNA helicase (FBH1) acting on the DNA structures resembling a stalled or collapsed replication fork and its interactions with RAD51 nucleoprotein filament. Individual helicase molecules isolated from human cells with their native post-translational modifications were analyzed using total internal reflection fluorescence microscopy. Separation of the activity trajectories originated from ubiquitylated and non-ubiquitylated FBH1 molecules revealed that ubiquitylation affects FBH1 interaction with the RAD51 nucleoprotein filament, but not its translocase and helicase activities.
doi:10.1093/nar/gkt056
PMCID: PMC3616717  PMID: 23393192
3.  Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures 
Nucleic Acids Research  2011;40(5):2089-2106.
Saccharomyces cerevisiae Mph1 is a 3–5′ DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3′ to 5′ directionality, Mph1 displaced 3′-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5′-flap strand more efficiently than the 3′ flap strand from double-flap substrates, which is not expected for a 3–5′ DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5′ single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.
doi:10.1093/nar/gkr983
PMCID: PMC3300029  PMID: 22090425
4.  Genetic and functional interactions between Mus81–Mms4 and Rad27 
Nucleic Acids Research  2010;38(21):7611-7625.
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.
doi:10.1093/nar/gkq651
PMCID: PMC2995070  PMID: 20660481
5.  Involvement of Vts1, a structure-specific RNA-binding protein, in Okazaki fragment processing in yeast 
Nucleic Acids Research  2009;38(5):1583-1595.
The non-essential VTS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes a sequence- and structure-specific RNA-binding protein. The Vts1 protein has been implicated in post-transcriptional regulation of a specific set of mRNAs that contains its-binding site at their 3′-untranslated region. In this study, we identified VTS1 as a multi-copy suppressor of dna2-K1080E, a lethal mutant allele of DNA2 that lacks DNA helicase activity. The suppression was allele-specific, since overexpression of Vts1 did not suppress the temperature-dependent growth defects of dna2Δ405N devoid of the N-terminal 405-amino-acid residues. Purified recombinant Vts1 stimulated the endonuclease activity of wild-type Dna2, but not the endonuclease activity of Dna2Δ405N, indicating that the activation requires the N-terminal domain of Dna2. Stimulation of Dna2 endonuclease activity by Vts1 appeared to be the direct cause of suppression, since the multi-copy expression of Dna2-K1080E suppressed the lethality observed with its single-copy expression. We found that vts1Δ dna2Δ405N and vts1Δdna2-7 double mutant cells displayed synergistic growth defects, in support of a functional interaction between two genes. Our results provide both in vivo and in vitro evidence that Vts1 is involved in lagging strand synthesis by modulating the Dna2 endonuclease activity that plays an essential role in Okazaki fragment processing.
doi:10.1093/nar/gkp1135
PMCID: PMC2836565  PMID: 20007605
6.  The Human F-Box DNA Helicase FBH1 Faces Saccharomyces cerevisiae Srs2 and Postreplication Repair Pathway Roles▿  
Molecular and Cellular Biology  2007;27(21):7439-7450.
The Saccharomyces cerevisiae Srs2 UvrD DNA helicase controls genome integrity by preventing unscheduled recombination events. While Srs2 orthologues have been identified in prokaryotic and lower eukaryotic organisms, human orthologues of Srs2 have not been described so far. We found that the human F-box DNA helicase hFBH1 suppresses specific recombination defects of S. cerevisiae srs2 mutants, consistent with the finding that the helicase domain of hFBH1 is highly conserved with that of Srs2. Surprisingly, hFBH1 in the absence of SRS2 also suppresses the DNA damage sensitivity caused by inactivation of postreplication repair-dependent functions leading to PCNA ubiquitylation. The F-box domain of hFBH1, which is not present in Srs2, is crucial for hFBH1 functions in substituting for Srs2 and postreplication repair factors. Furthermore, our findings indicate that an intact F-box domain, acting as an SCF ubiquitin ligase, is required for the DNA damage-induced degradation of hFBH1 itself. Overall, our findings suggest that the hFBH1 helicase is a functional human orthologue of budding yeast Srs2 that also possesses self-regulation properties necessary to execute its recombination functions.
doi:10.1128/MCB.00963-07
PMCID: PMC2169053  PMID: 17724085
7.  Diversity of swine Bordetella bronchiseptica isolates evaluated by RAPD analysis and PFGE 
Journal of Veterinary Science  2007;8(1):65-73.
The degree of genetic diversity in 45 Bordetella (B.) bronchiseptica strains comprised of a vaccine strain (N = 1), reference strains (N = 3) and field isolates (N = 41) was evaluated using random amplified polymorphic DNA (RAPD) fingerprinting and pulsed-field gel electrophoresis (PFGE). Three candidate primers were selected for RAPD analysis after screening 20 random decamer oligonucleotides for their discriminatory abilities. The OPA-07, OPA-08 and OPA-18 primers yielded 10, 10, and 6 distinct fingerprint patterns, respectively. The most common identical RAPD pattern was produced by OPA-07 which was shared by 32 isolates (71.1%), the pattern produced by OPA-08 was shared by 26 isolates (57.8%), and the pattern produced by OPA-18 was shared by 40 isolates (88.9%). The RAPD patterns of the vaccine strain and the 3 reference strains did not match any of the patterns produced by the field isolates when primers OPA-07 and OPA-08 were used. PFGE using the restriction endonuclease XbaI produced a total of 15 patterns consisting of 4 PFGE types (A, B, B1 and C, differing by ≥ 4 bands) and 11 A subtypes (differing by ≤ 3 bands). Most of the field isolates exhibited identical type A and B patterns, suggesting that they were related. The vaccine strain and the three reference strains showed different PFGE patterns as compared to the identical type A strains.
doi:10.4142/jvs.2007.8.1.65
PMCID: PMC2872699  PMID: 17322776
Bordetella bronchiseptica; genetic diversity; PFGE; RAPD
8.  Isolation of human Dna2 endonuclease and characterization of its enzymatic properties 
Nucleic Acids Research  2006;34(6):1854-1864.
In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase δ-catalyzed displacement DNA synthesis during Okazaki fragment processing depends on the combined action of Fen1 and Dna2. These two enzymes contain partially overlapping but distinct endonuclease activities. Dna2 is well-suited to process long flaps, which are converted to nicks by the subsequent action of Fen1. In this report, we purified human Dna2 as a recombinant protein from human cells transfected with the cDNA of the human homologue of Saccharomyces cerevisiae Dna2. We demonstrated that the purified human Dna2 enzyme contains intrinsic endonuclease and DNA-dependent ATPase activities, but is devoid of detectable DNA helicase activity. We determined a number of enzymatic properties of human Dna2 including its substrate specificity. When both 5′ and 3′ tailed ssDNAs were present in a substrate, such as a forked-structured one, both single-stranded regions were cleaved by human Dna2 (hDna2) with equal efficiency. Based on this and other properties of hDna2, it is likely that this enzyme facilitates the removal of 5′ and 3′ regions in equilibrating flaps that are likely to arise during the processing of Okazaki fragments in human cells.
doi:10.1093/nar/gkl102
PMCID: PMC1428795  PMID: 16595799
9.  In vivo and in vitro studies of Mgs1 suggest a link between genome instability and Okazaki fragment processing 
Nucleic Acids Research  2005;33(19):6137-6150.
The non-essential MGS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes an enzyme containing both DNA-dependent ATPase and DNA annealing activities. MGS1 appears to function in post-replicational repair processes that contribute to genome stability. In this study, we identified MGS1 as a multicopy suppressor of the temperature-sensitive dna2Δ405N mutation, a DNA2 allele lacking the N-terminal 405 amino acid residues. Mgs1 stimulates the structure-specific nuclease activity of Rad27 (yeast Fen1 or yFen1) in an ATP-dependent manner. ATP binding but not hydrolysis was sufficient for the stimulatory effect of Mgs1, since non-hydrolyzable ATP analogs are as effective as ATP. Suppression of the temperature-sensitive growth defect of dna2Δ405N required the presence of a functional copy of RAD27, indicating that Mgs1 suppressed the dna2Δ405N mutation by increasing the activity of yFen1 (Rad27) in vivo. Our results provide in vivo and in vitro evidence that Mgs1 is involved in Okazaki fragment processing by modulating Fen1 activity. The data presented raise the possibility that the absence of MGS1 may impair the processing of Okazaki fragments, leading to genomic instability.
doi:10.1093/nar/gki900
PMCID: PMC1275582  PMID: 16251400
10.  Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System 
The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.
doi:10.1128/AEM.71.7.3468-3474.2005
PMCID: PMC1169004  PMID: 16000750
11.  Use of Pseudomonas putida EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of Escherichia coli 
Applied and Environmental Microbiology  2004;70(12):6968-6976.
The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme β-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting. The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of E. coli. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.
doi:10.1128/AEM.70.12.6968-6976.2004
PMCID: PMC535197  PMID: 15574889
12.  Enzymatic properties of the Caenorhabditis elegans Dna2 endonuclease/helicase and a species-specific interaction between RPA and Dna2 
Nucleic Acids Research  2005;33(4):1372-1383.
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.
doi:10.1093/nar/gki255
PMCID: PMC552954  PMID: 15745997
13.  Genetics of lagging strand DNA synthesis and maturation in fission yeast: suppression analysis links the Dna2–Cdc24 complex to DNA polymerase δ 
Nucleic Acids Research  2004;32(21):6367-6377.
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.
doi:10.1093/nar/gkh963
PMCID: PMC535672  PMID: 15576681
14.  Fission Yeast Dna2 Is Required for Generation of the Telomeric Single-Strand Overhang 
Molecular and Cellular Biology  2004;24(21):9557-9567.
It has been suggested that the Schizosaccharomyces pombe Rad50 (Rad50-Rad32-Nbs1) complex is required for the resection of the C-rich strand at telomere ends in taz1-d cells. However, the nuclease-deficient Rad32-D25A mutant can still resect the C-rich strand, suggesting the existence of a nuclease that resects the C-rich strand. Here, we demonstrate that a taz1-d dna2-2C double mutant lost the G-rich overhang at a semipermissive temperature. The amount of G-rich overhang in S phase in the dna2-C2 mutant was lower than that in wild-type cells at the semipermissive temperature. Dna2 bound to telomere DNA in a chromatin immunoprecipitation assay. Moreover, telomere length decreased with each generation after shift of the dna2-2C mutant to the semipermissive temperature. These results suggest that Dna2 is involved in the generation of G-rich overhangs in both wild-type cells and taz1-d cells. The dna2-C2 mutant was not gamma ray sensitive at the semipermissive temperature, suggesting that the ability to process double-strand break (DSB) ends was not affected in the dna2-C2 mutant. Our results reveal that DSB ends and telomere ends are processed by different mechanisms.
doi:10.1128/MCB.24.21.9557-9567.2004
PMCID: PMC522233  PMID: 15485922
15.  Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast 
Nucleic Acids Research  2004;32(14):4205-4216.
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.
doi:10.1093/nar/gkh720
PMCID: PMC514361  PMID: 15302919
16.  SCFhFBH1 can act as helicase and E3 ubiquitin ligase 
Nucleic Acids Research  2004;32(8):2287-2297.
In our previous study, we found that a human F-box DNA helicase, named hFBH1, interacted with SKP1 to form an SCF (SKP1–Cul1–F-box protein) complex together with CUL1 and ROC1 in an F-box-dependent manner. The complex immunoprecipitated from crude cell extracts catalyzed polyubiquitin formation in the presence of the ubiquitin-activating and ubiquitin-conjugating enzymes, E1 and E2, respectively. In this report, we characterized the enzymatic properties of the recombinant SCFhFBH1 complex purified from insect cells expressing hFBH1, SKP1, CUL1 and ROC1. The SCFhFBH1 complex was isolated as a single tight complex that retained DNA helicase, DNA-dependent ATPase and E3 ubiquitin ligase activities. The helicase and ATPase activities residing in the SCFhFBH1 complex were indistinguishable from those of the hFBH1 protein alone. Moreover, the ubiquitin ligase activity of the SCFhFBH1 complex was hardly affected by single-stranded or double-stranded DNA. The multiple activities present in this complex act independently of each other, suggesting that the SCFhFBH1 complex can catalyze a ubiquitination reaction while acting as a DNA helicase or translocating along DNA. The potential roles of the SCFhFBH1 complex in DNA metabolism based upon the enzymatic activities associated with this complex are discussed.
doi:10.1093/nar/gkh534
PMCID: PMC419438  PMID: 15118074
17.  Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro 
Nucleic Acids Research  2003;31(12):3006-3015.
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.
PMCID: PMC162255  PMID: 12799426
18.  The fission yeast pfh1+ gene encodes an essential 5′ to 3′ DNA helicase required for the completion of S-phase 
Nucleic Acids Research  2002;30(21):4728-4739.
The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease–helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1+, encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5′ to 3′ DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the comple tion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.
PMCID: PMC135800  PMID: 12409464
19.  Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease 
Nucleic Acids Research  2001;29(14):3069-3079.
In order to gain insights into the structural basis of the multifunctional Dna2 enzyme involved in Okazaki fragment processing, we performed biochemical, biophysical and genetic studies to dissect the domain structure of Dna2. Proteolytic digestion of Dna2 using subtilisin produced a 127 kDa polypeptide that lacked the 45 kDa N-terminal region of Dna2. Further digestion generated two subtilisin-resistant core fragments of approximately equal size, 58 and 60 kDa. Surprisingly, digestion resulted in a significant (3- to 8-fold) increase in both ATPase and endonuclease activities compared to the intact enzyme. However, cells with a mutant DNA2 allele lacking the corresponding N-terminal region were severely impaired in growth, being unable to grow at 37°C, indicating that the N-terminal region contains a domain critical for a cellular function(s) of Dna2. Analyses of the hydrodynamic properties of and in vivo complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa domain revealed that Dna2 is active as the monomer and thus the defect in the mutant Dna2 protein is not due to its inability to multimerize. In addition, we found that the N-terminal 45 kDa domain interacts physically with a central region located between the two catalytic domains. Our results suggest that the N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the enzymatic activities of Dna2 by serving as a site for intra- and intermolecular interactions essential for optimal function of Dna2 in Okazaki fragment processing. The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing.
PMCID: PMC55803  PMID: 11452032
20.  The endonuclease activity of the yeast Dna2 enzyme is essential in vivo 
Nucleic Acids Research  2000;28(15):2873-2881.
Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activity well suited to remove RNA–DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 function. Systematic site-directed mutations of amino acid residues in Saccharomyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; (ii) inviable only when expression was repressed; (iii) inviable. Biochemical analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but were impaired in their endonuclease activity. Cells expressing mutant Dna2 enzymes partially impaired in endonuclease activity were viable, but were unable to grow when expression of their mutant Dna2 enzymes was further reduced. Their growth was restored when the mutant Dna2 proteins decreased in nuclease activity were induced to overexpress. In contrast, mutant Dna2 proteins lacking endonuclease activity did not allow cells to grow under any conditions tested. These in vivo and in vitro results demonstrate that the endonuclease activity of Dna2 is essential for Okazaki fragment processing.
PMCID: PMC102684  PMID: 10908349

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