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Juhani E. Syväoja - One of the best experts on this subject based on the ideXlab platform.
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mutations polymorphisms in the 55 kda subunit of DNA Polymerase Epsilon in human colorectal cancer
Cancer Genomics & Proteomics, 2009Co-Authors: Qi Zhou, Juhani E. Syväoja, Helmut Pospiech, Kati Talvinen, Jari Sundstrom, Adem Elzagheid, Yrjo CollanAbstract:Background: Defects of some DNA Polymerases have shown associations with cancer, but data on DNA Polymerase e are limited. This study investigated mutations in the 55 kDa subunit gene of DNA Polymerase e in colorectal cancer. Materials and Methods: DNA from 16 human colorectal cancer and 9 control samples was studied with Polymerase chain reaction-single-strand comformation polymorphism analysis and DNA sequencing. Results: DNA Polymerase e gene alterations were identified in 5 out of the 16 cases (31.2%). Two samples showed a T-C transition at exon 17 (potential tyrosine to histidine substitution), and an A-G transition at intron 7; one sample showed an A-G transition at intron 8. An AATT deletion was observed at intron 18 in 3 out of the 16 colon cancer cases (grades 2, 3, and 2, and Dukes' classes C, D, and C, respectively). Conclusion: Because the AATT deletion has also been found in breast cancer, the region may be a mutation hot spot, possibly involved in the carcinogenetic path in advanced colorectal cancer. The modified Dukes' class (or TNM classification) is the best general prognosticator for colon cancer (1). However, there are still situations in which other prognosticators will be able to offer valuable advice for clinicians, e.g. within individual classes. This especially applies to Dukes' class B cases, and proposals for additional prognosticators have been suggested (2). Many potential and proven prognostic features are negatively or positively associated with proliferation-
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genomic changes of the 55 kda subunit of DNA Polymerase Epsilon in human breast cancer
Cancer Genomics & Proteomics, 2008Co-Authors: Qi Zhou, Juhani E. Syväoja, Helmut Pospiech, Reza Effati, Kati Talvinen, Yrjo CollanAbstract:Background: DNA Polymerases (Pols) represent potential candidates for cancer genes because of their central functions in DNA metabolism. Defects of some DNA Pols have shown cancer associations, but data on DNA Polymerase (Pol) e is limited. Material s and Methods: Twenty-four human breast cancer DNA samples and four control DNA samples were examined for possible mutation in the entire coding region of the 55 kDa small subunit of the human DNA Pol e gene using Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the DNA and sequence analysis. In addition, 20 control DNAs were studied with PCR- SSCP for the end of intron 18 and exon 19 region. Results: An AATT deletion was found at one location in intron 18 in 2 out of the 24 breast cancer cases (8% ), but in none of the control cases. In addition, a single base transition was found in the cancer DNAs in intron 14, but the same changes were also found in the control DNAs, suggesting polymorphism. Conclusion:
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a neutralizing antibody against human DNA Polymerase Epsilon inhibits cellular but not sv40 DNA replication
Nucleic Acids Research, 1999Co-Authors: Helmut Pospiech, Juhani E. Syväoja, Inari Kursula, Waleed Abdelaziz, Linda H Malkas, Lahja Uitto, Maaret Kastelli, Maija Vihinenranta, Sinikka EskelinenAbstract:The contribution of human DNA Polymerase Epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA Polymerase activity of human DNA Polymerase Epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA Polymerase alpha. Contrary to the antibody against DNA Polymerase alpha, antibody K18 against DNA Polymerase Epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA Polymerase Epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA Polymerase alpha, and that this role for DNA Polymerase Epsilon cannot be modeled by SV40 DNA replication.
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cDNA and structural organization of the gene pole1 for the mouse DNA Polymerase Epsilon catalytic subunit
Biochimica et Biophysica Acta, 1999Co-Authors: Deqi Huang, Juhani E. Syväoja, Helmut Pospiech, Riikka Knuuti, H PalosaariAbstract:Abstract The cDNA and the gene for the mouse DNA Polymerase ϵ catalytic subunit were cloned. The deduced protein sequence shows remarkable evolutionary conservation in DNA Polymerase ϵ family. However, several conserved elements involved in template–primer binding differ from those of other class B Polymerases. This is likely to reflect a distinctive function of the enzyme. The gene that was assigned to chromosome 5 region E3–E5, consists of 49 exons and has a non-conforming splice site in the junction of exon and intron 13. A CpG island covers the promoter region which contains several putative consensus elements critical for S phase upregulated and serum responsive promoters.
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structural organization and splice variants of the pole1 gene encoding the catalytic subunit of human DNA Polymerase Epsilon
Biochemical Journal, 1999Co-Authors: Deqi Huang, T Kesti, Helmut Pospiech, Juhani E. SyväojaAbstract:The catalytic subunit of human DNA Polymerase Epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters.
Erik Johansson - One of the best experts on this subject based on the ideXlab platform.
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structural consequence of the most frequently recurring cancer associated substitution in DNA Polymerase Epsilon
Nature Communications, 2019Co-Authors: Vimal Parkash, Yashraj Kulkarni, Josy Ter Beek, Polina V Shcherbakova, Shina Caroline Lynn Kamerlin, Erik JohanssonAbstract:The most frequently recurring cancer-associated DNA Polymerase Epsilon (Pol Epsilon) mutation is a P286R substitution in the exonuclease domain. While originally proposed to increase genome instabi ...
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DNA Polymerase Epsilon bypasses an abasic site in the absence of a processivity clamp
2008Co-Authors: Nasim Sabouri, Erik JohanssonAbstract:To transfer the information in the genome from mother cell to daughter cell, the DNA replication must be carried out only once and with very high fidelity prior to every cell division. In yeast there are several different DNA Polymerases involved in DNA replication and/or DNA repair. The two replicative DNA Polymerases, DNA Polymerase delta (Pol delta) and DNA Polymerase Epsilon (Pol Epsilon), which both include a proofreading 3´→5´exonuclease activity, can replicate and proofread the genome with a very high degree of accuracy. The aim of this thesis was to gain a better understanding of how the enigmatic DNA Polymerase Epsilon participates in DNA transactions. To investigate whether Pol Epsilon or Pol delta is responsible for the synthesis of DNA on the lagging strand, the processing and assembly of Okazaki fragments was studied. Pol delta was found to have a unique property called “idling” which, together with the flap-endonuclease (FEN1), maintained a ligatable nick for DNA ligase I. In contrast, Pol Epsilon was found to lack the ability to “idle” and interact functionally with FEN-1, indicating that Pol Epsilon is not involved in processing Okazaki fragments. Together with previous genetic studies, it was concluded that Pol delta is the preferred lagging strand Polymerase, leaving Pol Epsilon to carry out some other function. The structure of Pol Epsilon was determined by cryo-electron microscopy, to a resolution of ~20 A. Pol Epsilon is composed of a globular “head” domain consisting of the large catalytic subunit Pol2p, and a “tail” domain, consisting of the small subunits Dpb2p, Dpb3p, and Dpb4p. The two separable domains were found to be connected by a flexible hinge. Interestingly, the high intrinsic processivity of Pol Epsilon depends on the interaction between the tail domain and double-stranded DNA. As a replicative DNA Polymerase, Pol Epsilon encounters different lesions in DNA. It was shown that Pol Epsilon can perform translesion synthesis (TLS) through a model abasic site in the absence of external processivity clamps under single-hit conditions. The lesion bypass was dependent of the sequence on the template and also on a proper interaction of the “tail”domain with the primer-template. Yeast cells treated with a DNA damaging agent and devoid of all TLS Polymerases showed improved survival rates in the presence of elevated levels of dNTPs. These genetic results suggested that replicative Polymerases may be engaged in the bypass of some DNA lesions. In vitro, Pol Epsilon was found to bypass 8-OxoG at elevated dNTP levels. Together, the in vitro and in vivo results suggest that the replicative Polymerases may be engaged in bypass of less bulky DNA lesions at elevated dNTP levels. In conclusion, the low-resolution structure presented represents the first structural characterization of a eukaryotic multi-subunit DNA Polymerase. The replicative DNA Polymerase Pol Epsilon can perform translesion synthesis due to an interaction between the tail domain and double-stranded DNA. Pol Epsilon may also bypass less bulky DNA lesions when there are elevated dNTP concentrations in vivo.
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charecterization of a DNA Polymerase Epsilon complex lacking the essential subunit dpb2
2008Co-Authors: Isabelle Isoz, Kirill V Volkov, Erik JohanssonAbstract:Each cell division, the nuclear DNA must be replicated efficiently and with high accuracy to avoid mutations which can have an effect on cell function. There are three replicative DNA Polymerases essential for the synthesis of DNA during replication in eukaryotic cells. DNA Polymerase α (Pol α) synthesize short primers required for DNA Polymerase δ (Pol δ) and DNA Polymerase e (Pol e) to carry out the bulk synthesis. The role of Pol δ and Pol e at the replication fork has been unclear. The aim of this thesis was to examine what role Pol e has at the replication fork, compare the biochemical properties of Pol δ and Pol e, and to study the function of the second largest and essential subunit of Pol e, Dpb2. To identify where Pol e replicates DNA in vivo, a strategy was taken where the active site of Pol e was altered to create a mutator Polymerase leaving a unique error-signature. A series of mutant pol e proteins were purified and analyzed for enzyme activity and fidelity of DNA synthesis. Two mutants, M644F and M644G, exhibited an increased mutation rate and close to normal Polymerase activity. One of these, the M644G gave rise to a specific increase of mismatch mutations resulting from T-dTMP mis-pairing during DNA synthesis in vitro. The M644G mutant was introduced in yeast strains carrying a reporter gene, URA3, on either side of an origin in different orientations. Mutations which inactivated the URA3 gene in the M644G mutant strains were analyzed. A strand specific signature was found demonstrating that Pol e participates in the synthesis of the leading strand. Pol δ and Pol e are both stimulated by the processivity clamp, PCNA, in in vitro replication assays. To clarify any differences they were challenged side by side in biochemical assays. Pol e was found to require that single-stranded template (ssDNA) was entirely coated with RPA, whereas Pol δ was much less sensitive to uncoated ssDNA. The processivity of Pol δ was stimulated to a much higher degree by PCNA than of Pol e. In presence of PCNA the processivity of Pol δ and Pol e was comparable. In contrast, Pol e was approximately four times slower than Pol δ when replicating a single-primed circular template in the presence of all accessory proteins and an excess of Polymerase. The biochemical characterization of the system suggests that Pol e and Pol δ are loaded onto the PCNA-primer-ternary complex by separate mechanisms. A model is proposed where the loading of Pol e onto the leading strand is independent of the PCNA interaction motif which is required by enzymes acting on the lagging strand. The essential gene DPB2 encodes for the second largest subunit of Pol e. We carried out a genetic screen in S.cerevisiae and isolated a lethal mutant allele of dpb2 (dpb2-200). When over-expressed together with the remaining three subunits of Pole, Pol2, Dpb3 and Dpb4, the dpb2-201 did not copurify. The biochemical property of Pol2/Dpb3/Dpb4 complex was compared with wild-type four-subunit Pol e (Pol2/Dpb2/Dpb3/Dpb4) and a Pol2/Dpb2 complex in replication assays. The absence of Dpb2 in the complex did not significantly affect the specific activity or the processivity, but gave a slightly reduced efficiency in holoenzyme assays when compared to wild-type four-subunit Pol e. We propose that Dpb2 is not essential for the enzyme activity of Pol e.
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The eukaryotic leading and lagging strand DNA Polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA
Nucleic Acids Research, 2007Co-Authors: Olga Chilkova, Peter Stenlund, Isabelle Isoz, Pawel Grabowski, Else-britt Lundström, Carrie M Stith, Peter M J Burgers, Erik JohanssonAbstract:Saccharomyces cerevisiae DNA Polymerase delta (Pol delta) and DNA Polymerase Epsilon (Pol Epsilon) are replicative DNA Polymerases at the replication fork. Both enzymes are stimulated by PCNA, alth ...
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regulation of b family DNA Polymerase fidelity by a conserved active site residue characterization of m644w m644l and m644f mutants of yeast DNA Polymerase e
Nucleic Acids Research, 2007Co-Authors: Zachary F Pursell, Isabelle Isoz, Else-britt Lundström, Erik Johansson, Thomas A KunkelAbstract:Regulation of B family DNA Polymerase fidelity by a conserved active site residue : characterization of M644W, M644L and M644F mutants of yeast DNA Polymerase Epsilon.
T Kesti - One of the best experts on this subject based on the ideXlab platform.
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cell cycle dependent phosphorylation of the DNA Polymerase Epsilon subunit dpb2 by the cdc28 cyclin dependent protein kinase
Journal of Biological Chemistry, 2004Co-Authors: T Kesti, Hayes W Mcdonald, John R Yates, Curt WittenbergAbstract:DNA Polymerase ϵ (Polϵ), one of the three major eukaryotic replicative Polymerases, is comprised of the essential catalytic subunit, called Pol2 in budding yeast, and three accessory subunits, only one of which, Dpb2, is essential. Polϵ is recruited to replication origins during late G1 phase prior to activation of replication. In this work we show that the budding yeast Dpb2 is phosphorylated in a cell cycle-dependent manner during late G1 phase. Phosphorylation results in the appearance of a lower mobility species. The appearance of that species in vivo is dependent upon the Cdc28 cyclin-dependent protein kinase (CDK), which can directly phosphorylate Dpb2 in vitro. Either G1 cyclin (Cln) or B-type cyclin (Clb)-associated CDK is sufficient for phosphorylation. Mapping of phosphorylation sites by mass spectrometry using a novel gel-based proteolysis protocol shows that, of the three consensus CDK phosphorylation sites, at least two, Ser-144 and Ser-616, are phosphorylated in vivo. The Cdc28 CDK phosphorylates only Ser-144 in vitro. Using site-directed mutagenesis, we show that Ser-144 is sufficient for the formation of the lower mobility form of Dpb2 in vivo. In contrast, Ser-616 appears not to be phosphorylated by Cdc28. Finally, inactivation of all three CDK consensus sites in Dpb2 results in a synthetic phenotype with the pol2-11 mutation, leading to decreased spore viability, slow growth, and increased thermosensitivity. We suggest that phosphorylation of Dpb2 during late G1 phase at CDK consensus sites facilitates the interaction with Pol2 or the activity of Polϵ
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structural organization and splice variants of the pole1 gene encoding the catalytic subunit of human DNA Polymerase Epsilon
Biochemical Journal, 1999Co-Authors: Deqi Huang, T Kesti, Helmut Pospiech, Juhani E. SyväojaAbstract:The catalytic subunit of human DNA Polymerase Epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters.
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DNA Polymerase e catalytic domains are dispensable for DNA replication DNA repair and cell viability
Molecular Cell, 1999Co-Authors: Juhani E. Syväoja, T Kesti, Karin Flick, Sirkka Keranen, Curt WittenbergAbstract:DNA Polymerase Epsilon (Pol Epsilon) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol Epsilon is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion of budding yeast Pol Epsilon (Pol2) containing all known DNA Polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability. However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability. Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.
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localization of the gene for DNA Polymerase Epsilon pole to human chromosome 12q24 3 and rat chromosome 12 by somatic cell hybrid panels and fluorescence in situ hybridization
Genomics, 1994Co-Authors: Josiane Szpirer, Juhani E. Syväoja, T Kesti, Florence Pedeutour, Michele Riviere, Claude Turccarel, Claude SzpirerAbstract:DNA Polymerase Epsilon [DNA pol Epsilon (EC 2.7.7.7)] is one of the four nuclear DNA Polymerases in eukaryotic cells. The mammalian enzyme is involved in DNA repair and possibly also in replication of chromosomal DNA. The gene encoding pol Epsilon (POLE) was assigned to human and rat chromosomes 12 by Southern blot analysis of genomic DNA from mouse-human and mouse-rat somatic cell hybrid panels using human cDNA probe. The human gene was then regionally localized to band 12q24.3 by fluorescence in situ hybridization of metaphase spreads of chromosomes from human lymphocytes using genomic DNA probe. POLE is closely linked to HNF1A, a gene encoding a liver-enriched transcription factor, HNF1 alpha. The two genes thus define a new synteny group retained on human and rat chromosomes 12. Another gene mapping to the same or close-by region as human POLE is a gene for inherited disorder tuberous sclerosis 3, TSC3.
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molecular cloning of the cDNA for the catalytic subunit of human DNA Polymerase Epsilon
Journal of Biological Chemistry, 1993Co-Authors: T Kesti, H Frantti, J E SyvaojaAbstract:The cDNA encoding the catalytic polypeptide of human DNA Polymerase Epsilon was cloned. The deduced amino acid sequence reveals that the catalytic polypeptide is 2257 amino acids in length and its calculated molecular mass is 258 kDa. A single RNA message of 7.5 kilobases was recognized by isolated cDNA clones. The identity of the cDNA was verified by direct amino acid sequencing of tryptic fragments derived from the catalytic polypeptide of the HeLa DNA Polymerase Epsilon. The primary structure comparison with multiple DNA Polymerases indicates that human DNA Polymerase Epsilon catalytic polypeptide is a homolog of the yeast Saccharomyces cerevisiae DNA Polymerase II catalytic polypeptide. The proteins are 39% identical. In the region containing known DNA Polymerase consensus motifs, the identity is 63%. The expression of the mRNA encoding DNA Polymerase Epsilon is strongly dependent on cell proliferation.
Judith L Campbell - One of the best experts on this subject based on the ideXlab platform.
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mutations in the non catalytic subunit dpb2 of DNA Polymerase Epsilon affect the nrm1 branch of the DNA replication checkpoint
PLOS Genetics, 2017Co-Authors: Michal Dmowski, Piotr Jonczyk, Judith L Campbell, Justyna Rudzka, Iwona J FijalkowskaAbstract:To preserve genome integrity, the S-phase checkpoint senses damaged DNA or nucleotide depletion and when necessary, arrests replication progression and delays cell division. Previous studies, based on two pol2 mutants have suggested the involvement of DNA Polymerase Epsilon (Pol e) in sensing DNA replication accuracy in Saccharomyces cerevisiae. Here we have studied the involvement of Pol e in sensing proper progression of DNA replication, using a mutant in DPB2, the gene coding for a non-catalytic subunit of Pol e. Under genotoxic conditions, the dpb2-103 cells progress through S phase faster than wild-type cells. Moreover, the Nrm1-dependent branch of the checkpoint, which regulates the expression of many replication checkpoint genes, is impaired in dpb2-103 cells. Finally, deletion of DDC1 in the dpb2-103 mutant is lethal supporting a model of strand-specific activation of the replication checkpoint. This lethality is suppressed by NRM1 deletion. We postulate that improper activation of the Nrm1-branch may explain inefficient replication checkpoint activation in Pol e mutants.
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defective interaction between pol2p and dpb2p subunits of DNA Polymerase Epsilon contributes to a mutator phenotype in saccharomyces cerevisiae
Mutation Research, 2009Co-Authors: Malgorzata Jaszczur, Krzysztof Flis, Iwona J Fijalkowska, Judith L Campbell, Justyna Rudzka, Joanna Kraszewska, Piotr Polaczek, Piotr JonczykAbstract:Most of the prokaryotic and eukaryotic replicative Polymerases are multi-subunit complexes. There are several examples indicating that noncatalytic subunits of DNA Polymerases may function as fidelity factors during replication process. In this work, we have further investigated the role of Dpb2p, a noncatalytic subunit of DNA Polymerase Epsilon holoenzyme from Saccharomyces cerevisiae in controlling the level of spontaneous mutagenesis. The data presented indicate that impaired interaction between catalytic Pol2p subunit and Dpb2p is responsible for the observed mutator phenotype in S. cerevisiae strains carrying different mutated alleles of the DPB2 gene. We observed a significant correlation between the decreased level of interaction between different mutated forms of Dpb2p towards a wild-type form of Pol2p and the strength of mutator phenotype that they confer. We propose that structural integrity of the Pol Epsilon holoenzyme is essential for genetic stability in S. cerevisiae cells.
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mrc1 and DNA Polymerase Epsilon function together in linking DNA replication and the s phase checkpoint
Molecular Cell, 2008Co-Authors: Huiqiang Lou, Martin E Budd, Makiko Komata, Yuki Katou, Zhiyun Guan, Clara C Reis, Katsuhiko Shirahige, Judith L CampbellAbstract:Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA Polymerase Є, essential for leading-strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding, but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replication forks stalled in HILI. The bimodal Mrc1/Pol2 interaction may be an additional step in regulating the S phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, which also interacts with the MCMs, may modulate coupling of polymerization and unwinding at the replication fork.
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dpb2p a noncatalytic subunit of DNA Polymerase Epsilon contributes to the fidelity of DNA replication in saccharomyces cerevisiae
Genetics, 2008Co-Authors: Malgorzata Jaszczur, Krzysztof Flis, Piotr Jonczyk, Judith L Campbell, Justyna Rudzka, Joanna Kraszewska, Piotr Polaczek, Martin E Budd, Iwona J FijalkowskaAbstract:Most replicases are multi-subunit complexes. DNA Polymerase Epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 3′ → 5′ proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA Polymerase subunits other than those housing the DNA Polymerase and 3′ → 5′ exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.
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in vivo reconstitution of saccharomyces cerevisiae DNA Polymerase Epsilon in insect cells purification and characterization
Journal of Biological Chemistry, 2002Co-Authors: Rajiv Dua, Daniel Levy, Peter M Snow, Judith L CampbellAbstract:DNA Polymerase Epsilon (pol Epsilon) is a multiple subunit complex consisting of at least four proteins, including catalytic Po12p, Dpb2p, Dpb3p, and Dpb4p. Pol Epsilon has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol Epsilon complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol Epsilon complex and truncated Po12p (140 kDa), as was observed initially with the pol Epsilon purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol Epsilon is functionally identical to native pol Epsilon purified from yeast and is therefore useful for biochemical characterization of the interactions of pol Epsilon with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Po12p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Po12p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc.
Akio Sugino - One of the best experts on this subject based on the ideXlab platform.
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double stranded DNA binding properties of saccharomyces cerevisiae DNA Polymerase Epsilon and of the dpb3p dpb4p subassembly
Genes to Cells, 2003Co-Authors: Toshiaki Tsubota, Akio Sugino, Satoko Maki, Hajime Kubota, Hisaji MakiAbstract:Background: DNA Polymerase ɛ (Pol ɛ) of Saccharomyces cerevisiae participates in many aspects of DNA replication, as well as in DNA repair. In order to clarify molecular mechanisms employed in the multiple tasks of Pol ɛ, we have been characterizing the interaction between Pol ɛ and DNA. Results: Analysis of the four-subunit Pol ɛ complex by gel mobility shift assay revealed that the complex binds not only to single-stranded (ss) DNA but also equally well to double-stranded (ds) DNA. A truncated polypeptide consisting of the N-terminal domain of Pol2p catalytic subunit binds to ssDNA but not to dsDNA, indicating that the Pol2p C-terminal domain and/or the auxiliary subunits are involved in the dsDNA-binding. The dsDNA-binding by Pol ɛ does not require DNA ends or specific DNA sequences. Further analysis by competition experiments indicated that Pol ɛ contains at least two distinct DNA-binding sites, one of which binds exclusively to ssDNA and the other to dsDNA. The dsDNA-binding site, however, is suggested to also bind ssDNA. The DNA Polymerase activity of Pol ɛ is inhibited by ssDNA but not by dsDNA. Furthermore, purification of the Pol ɛ auxiliary subunits Dpb3p and Dpb4p revealed that these proteins form a heterodimer and associate with dsDNA. Conclusions: Pol ɛ has multiple sites at which it interacts with DNA. One of these sites has a strong affinity for dsDNA, a feature that is not generally associated with DNA Polymerases. Involvement of the Dpb3p-Dpb4p complex in the dsDNA-binding of Pol ɛ is inferred.
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DNA Polymerase Epsilon is required for coordinated and efficient chromosomal DNA replication in xenopus egg extracts
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Shou Waga, Taro Masuda, Haruhiko Takisawa, Akio SuginoAbstract:Abstract DNA Polymerase ɛ (Polɛ) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA Polymerases, DNA Polymerases α and δ (Polα and δ), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Polɛ in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Polɛ is indeed required for chromosomal DNA replication. In Polɛ-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Polα are loaded onto the chromatin template in Polɛ-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Polɛ, along with Polα and Polδ, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.
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DNA Polymerase Epsilon encoded by cdc20 is required for chromosomal DNA replication in the fission yeast schizosaccharomyces pombe
Genes to Cells, 1998Co-Authors: Akio Sugino, Takeshi Ohara, Josef Sebastian, Naomi Nakashima, Hiroyuki ArakiAbstract:Background DNA Polymerase II (PolII), the homologue of mammalian DNA Polymerase Epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA Polymerase Epsilon. It has been shown that DNA Polymerase Epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts. Results We have cloned and sequenced S. pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA Polymerase Epsilon (Pol Epsilon). The predicted amino acid sequence of Pol Epsilon is highly homologous to that of S. cerevisiae PolII and human Pol Epsilon. Consistent with this, the Pol Epsilon polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Pol Epsilon activity from temperature-sensitive S. pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures. Conclusion These data strongly suggest that Pol Epsilon is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.
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DNA Polymerase ii the probable homolog of mammalian DNA Polymerase Epsilon replicates chromosomal DNA in the yeast saccharomyces cerevisiae
The EMBO Journal, 1992Co-Authors: Hiroyuki Araki, P Ropp, A L Johnson, L H Johnston, Alan Morrison, Akio SuginoAbstract:Abstract Two temperature-sensitive DNA Polymerase II mutants (pol2-9 and pol2-18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method. DNA Polymerase II activity partially purified from both mutants was thermolabile, while DNA Polymerase I and III activities remained thermotolerant. At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants. The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes. These results indicate that DNA Polymerase II participates in nuclear DNA replication. The similarities in structure and activities between the DNA Polymerases of yeast and mammals make it likely that mammalian DNA Polymerase Epsilon too is required for chromosomal DNA replication.