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1.
Nucleic Acids Res ; 34(6): 1854-64, 2006.
Article in English | MEDLINE | ID: mdl-16595799

ABSTRACT

In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase delta-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.


Subject(s)
DNA Helicases/metabolism , Endodeoxyribonucleases/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/isolation & purification , Adenosine Triphosphatases/metabolism , Cell Line , DNA/metabolism , DNA Helicases/genetics , DNA Helicases/isolation & purification , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/isolation & purification , Flap Endonucleases/metabolism , Humans , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Substrate Specificity
2.
Nucleic Acids Res ; 33(19): 6137-50, 2005.
Article in English | MEDLINE | ID: mdl-16251400

ABSTRACT

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 dna2Delta405N 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 dna2Delta405N required the presence of a functional copy of RAD27, indicating that Mgs1 suppressed the dna2Delta405N 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.


Subject(s)
Adenosine Triphosphatases/physiology , DNA Helicases/physiology , DNA/metabolism , Genomic Instability , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , Flap Endonucleases/metabolism , Genes, Suppressor , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Deletion
3.
Nucleic Acids Res ; 33(4): 1372-83, 2005.
Article in English | MEDLINE | ID: mdl-15745997

ABSTRACT

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 (approximately 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 delta-mediated displacement DNA synthesis are mostly short in C.elegans eukaryotes, and hence less dependent on Dna2 for viability.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/enzymology , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Adenosine Triphosphatases/metabolism , Animals , Caenorhabditis elegans Proteins/genetics , DNA Helicases/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , Endodeoxyribonucleases/genetics , RNA/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Replication Protein A , Saccharomyces cerevisiae Proteins/metabolism , Species Specificity
4.
Nucleic Acids Res ; 32(21): 6367-77, 2004.
Article in English | MEDLINE | ID: mdl-15576681

ABSTRACT

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 delta. 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.


Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/physiology , DNA Polymerase III/genetics , DNA Replication , Flap Endonucleases/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/physiology , Schizosaccharomyces/genetics , Alleles , Base Sequence , DNA Polymerase III/metabolism , DNA, Fungal/biosynthesis , Flap Endonucleases/metabolism , Genes, Fungal , Genetic Complementation Test , Molecular Sequence Data , Mutation , Phenotype , Proliferating Cell Nuclear Antigen/metabolism , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Suppression, Genetic
5.
Nucleic Acids Res ; 32(14): 4205-16, 2004.
Article in English | MEDLINE | ID: mdl-15302919

ABSTRACT

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 -mediated lagging strand displacement DNA synthesis.


Subject(s)
DNA Helicases/genetics , DNA Helicases/physiology , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/physiology , Schizosaccharomyces/enzymology , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/genetics , Cell Division , DNA/chemistry , DNA/metabolism , DNA Helicases/metabolism , Escherichia coli Proteins , Genes, Fungal , Mutation , Nucleic Acid Conformation , Peptide Elongation Factors/genetics , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/metabolism , Substrate Specificity , Suppression, Genetic , Templates, Genetic , Transcription Factors/genetics , Transcriptional Elongation Factors
6.
Nucleic Acids Res ; 32(8): 2287-97, 2004.
Article in English | MEDLINE | ID: mdl-15118074

ABSTRACT

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 SCF(hFBH1) complex purified from insect cells expressing hFBH1, SKP1, CUL1 and ROC1. The SCF(hFBH1) 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 SCF(hFBH1) complex were indistinguishable from those of the hFBH1 protein alone. Moreover, the ubiquitin ligase activity of the SCF(hFBH1) 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 SCF(hFBH1) complex can catalyze a ubiquitination reaction while acting as a DNA helicase or translocating along DNA. The potential roles of the SCF(hFBH1) complex in DNA metabolism based upon the enzymatic activities associated with this complex are discussed.


Subject(s)
DNA Helicases/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphatases/metabolism , DNA/pharmacology , DNA Helicases/genetics , DNA-Binding Proteins , Humans , Kinetics , Macromolecular Substances , Recombinant Proteins/metabolism , SKP Cullin F-Box Protein Ligases/genetics , Ubiquitins/metabolism
7.
Nucleic Acids Res ; 30(21): 4728-39, 2002 Nov 01.
Article in English | MEDLINE | ID: mdl-12409464

ABSTRACT

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 completion 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.


Subject(s)
DNA Helicases/metabolism , Genes, Essential/genetics , S Phase , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/enzymology , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/isolation & purification , Adenosine Triphosphatases/metabolism , Alleles , Catalysis , Cell Cycle Proteins/genetics , Cell Cycle Proteins/isolation & purification , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Conserved Sequence , DNA Damage/drug effects , DNA Helicases/genetics , DNA Helicases/isolation & purification , DNA Helicases/pharmacology , DNA Repair , DNA Replication , DNA, Fungal/analysis , G2 Phase , Gene Deletion , Genes, Fungal/genetics , Hydroxyurea/pharmacology , Methyl Methanesulfonate/pharmacology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Fungal/genetics , RNA, Fungal/metabolism , S Phase/drug effects , Schizosaccharomyces/drug effects , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/isolation & purification , Schizosaccharomyces pombe Proteins/pharmacology , Suppression, Genetic , Temperature
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