The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta.

Bauer, Glenn A.; Burgers, Peter M.

doi:10.1073/pnas.85.20.7506

Cited by 137 publications

(89 citation statements)

References 30 publications

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“…The enzyme is usually isolated as a heterodimer with subunits of approximately 125 and 50 kDa, although the Drosophila enzyme has a single subunit of 120 kDa (10) and both one-and two-subunit forms of pol ␦ were isolated from mouse cells (9). PCNA was found to stimulate the activity and processivity of heterodimeric forms of pol ␦ (8,9,11,12) but to have no effect on the isolated catalytic subunit (9,10). The catalytic (125 kDa) subunit has DNA polymerase and 3Ј-5Ј-exonuclease activity (13,14).…”

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confidence: 99%

“…pol ␦ has been purified from a number of eukaryotic sources including calf thymus (7), budding yeast (8), mouse cells (9), and Drosophila melanogaster (10). The enzyme is usually isolated as a heterodimer with subunits of approximately 125 and 50 kDa, although the Drosophila enzyme has a single subunit of 120 kDa (10) and both one-and two-subunit forms of pol ␦ were isolated from mouse cells (9).…”

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confidence: 99%

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Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

Zhou¹,

Tan

et al. 1996

Journal of Biological Chemistry

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The catalytic subunit of human DNA polymerase ␦ has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a M r ‫؍‬ ϳ125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase ␦. The recombinant protein was purified to near homogeneity (approximately 1200-fold) from insect cells by chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3 -5 exonuclease activities. The properties of the recombinant catalytic subunit were compared with those of the native heterodimeric DNA polymerase ␦ isolated from fetal calf thymus, and the enzymes were found to differ in several respects. Although the native heterodimer is equally active with either Mn 2؉ or Mg 2؉ as divalent cation activator, the recombinant catalytic subunit is approximately 5-fold more active in Mn 2؉ than in Mg 2؉ . The most striking difference between the two proteins is the response to the proliferating cell nuclear antigen (PCNA). The activity and processivity of native DNA polymerase ␦ are markedly stimulated by PCNA whereas it has no effect on the recombinant catalytic subunit. These results suggest that the small subunit of DNA polymerase ␦ is essential for functional interaction with PCNA.Although genetic studies in yeast have suggested that three distinct DNA polymerases (␣, ␦, and ⑀) are required for nuclear DNA replication in eukaryotes (1-4), only two DNA polymerases (␣ and ␦) were found to be essential components of an in vitro reconstituted SV40 DNA replication system (5); pol 1 ␣, with its tightly associated primase activity, was shown to be required for the synthesis of primers for both leading and lagging strands, whereas pol ␦ and its accessory proteins, proliferating cell nuclear antigen (PCNA) and replication factor C, also known as activator 1, were found to be required for leading strand synthesis and for completing Okazaki fragments initiated by pol ␣/primase. More recently, elucidation of the roles of DNA polymerases ␣, ␦, and ⑀ in DNA replication was approached by UV cross-linking of polymerases to nascent DNA within replicating chromosomes (6). These studies showed that only pol ␣ and pol ␦ were photolabeled by nascent SV40 DNA whereas pol ␣, pol ␦, and pol ⑀ were all photolabeled by nascent cellular DNA, suggesting that the replication of SV40 chromosomes may require only a subset of the proteins required for replication of cellular chromosomes. Thus, the roles of pol ␦ and pol ⑀ in cellular DNA replication may not be resolved by studies of the replication of DNA viruses; rather, studies of the properties of the replicative DNA polymerases and their interactions with other replication proteins will be necessary to answer these questions.pol ␦ has been purified from a number of eukaryotic sources including calf thymus (7), budding yeast (8), mouse cells (9), and Drosophila melanogaster (10). The enzyme is usually isol...

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Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

Zhou¹,

Tan

et al. 1996

Journal of Biological Chemistry

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“…This series of reactions leads to a polymerase switch in which the pol ␣-primase complex at primer ends is displaced and replaced by pol ␦. Okazaki fragments are then elongated by pol ␦ until they encounter downstream Okazaki fragments (2,(8)(9)(10). Pol ␦ continues to synthesize DNA by displacing the 5Ј termini of downstream Okazaki fragments, which generate 5Ј RNA-DNA flap structures (11).…”

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The MPH1 Gene of Saccharomyces cerevisiae Functions in Okazaki Fragment Processing

Kang

Kim

et al. 2009

Journal of Biological Chemistry

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Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3 to 5 DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the processing of Okazaki fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2⌬405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2⌬405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5 to 3 exonuclease activity of Fen1. Mph1 binds to flapstructured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2⌬405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating the formation of ligatable nicks.Lagging strand DNA synthesis requires the orchestrated actions of many proteins and can be divided into several distinct enzymatic steps (1-4). First, the polymerase (pol) 2 ␣-primase complex synthesizes RNA-DNA primers on the template DNA that are recognized by replication factor C. This complex loads proliferating cell nuclear antigen onto DNA, which acts as a processivity factor tethering pol ␦ to primer ends (5-7). This series of reactions leads to a polymerase switch in which the pol ␣-primase complex at primer ends is displaced and replaced by pol ␦. Okazaki fragments are then elongated by pol ␦ until they encounter downstream Okazaki fragments (2,(8)(9)(10). Pol ␦ continues to synthesize DNA by displacing the 5Ј termini of downstream Okazaki fragments, which generate 5Ј RNA-DNA flap structures (11). These flaps are then cleaved by structure-specific nucleases that lead to the generation of ligatable nicks, which are sealed by DNA ligase converting the noncontiguous lagging strands to a contiguous DNA chain (12, 13).

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“…Indeed, several reports demonstrate that PCNA trimeric structure is stable both on DNA and in solution. 44,45 Additionally, PCNA was previously detected as dimer and trimer on 2D SDS-PAGE 42 and, more recently, double trimeric structure was described. 31,32 Our findings therefore agree with those reported in the literature.…”

Section: Pcna Isoforms Expression In Hccmentioning

confidence: 99%

Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation

Venturi

Piaz

Giovannini

et al. 2008

Laboratory Investigation

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Proliferating cell nuclear antigen (PCNA) is a 36 kDa protein involved in several cellular mechanisms, including DNA synthesis and repair, cell cycle regulation and apoptosis. An alteration in PCNA structure might contribute to DNA-damage accumulation in cancer cells. This study was aimed to evaluate the PCNA pattern of expression, in terms of aggregation status, isoforms and post-translational modifications, in human hepatocellular carcinoma (HCC) and cirrhosis as well as in HCC cell lines. Twelve HCCs and surrounding cirrhotic tissues were analysed, along with HepG2, Hep3B and SNU-398 cell lines. Normal liver specimens and cirrhosis without HCC were included as controls. Both DNA-bound and DNA-unbound PCNA fractions were analysed, and PCNA pattern of expression was displayed on two-dimensional gel electrophoresis followed by western blot. Results were confirmed by mass spectrometry. To compare HCCs vs surrounding tissues, immunolabelling and immunostaining were performed. In 6 of 12 HCCs and in cell lines, we found three major PCNA acidic forms, corresponding to monomers, probably dimers and trimers, and a basic isoform. In the six remaining HCCs, only a PCNA acidic form associated with multiple basic isoforms was detected. Importantly, the PCNA basic form was not found in cirrhotic tissues. To clarify the nature of the detected PCNA isoforms, ubiquitin-specific immunoblotting as well as phosphatase treatment were employed. A PCNA-ubiquitylated form in cell lines and PCNA-phosphorylated isoforms in 6 of 12 HCCs were detected. Finally, in the DNA-bound fraction we detected only an acidic PCNA monomeric form. We conclude that human hepatocellular carcinoma expresses specific PCNA isoforms compared to those found in cirrhosis, implicating a role for PCNA functional alterations in hepatocarcinogenesis.

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The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta.

Cited by 137 publications

References 30 publications

Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

The MPH1 Gene of Saccharomyces cerevisiae Functions in Okazaki Fragment Processing

Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation

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