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Teresa S.-f. Wang - One of the best experts on this subject based on the ideXlab platform.

  • Interaction of herpes simplex virus 1 origin-binding protein with DNA Polymerase Alpha.
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Sam S. K. Lee, Qun Dong, Teresa S.-f. Wang, I. R. Lehman
    Abstract:

    Abstract The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA Polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA Polymerase Alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA Polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.

  • enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication
    Journal of Biological Chemistry, 1993
    Co-Authors: William C Copeland, Teresa S.-f. Wang
    Abstract:

    The enzymatic mechanism of primase was investigated using Escherichia coli and baculoviral overexpressed mouse primase subunits, p49 and p58. Neither of the singly purified primase subunits displayed primase activity alone, but the p49 subunit was able to extend a riboprimer, indicating that this subunit contains an RNA Polymerase activity. The p58 subunit cooperated with the p49 subunit in binding the initiating purine to form the initial dinucleotide. After initiation, the p49 subunit alone was sufficient to extend the growing primer, but both the rate of p49 primer extension and its stability were influenced by the p58 subunit. The Km(ATP) in primer synthesis on poly(dT) of the p49-p58 heterodimeric primase complex was 10-fold higher than the Km(ATP) of the single p49 subunit in a ribo(A) primer extension assay. In addition, labeled ATP cross-linked to both of the individually purified subunits but with a striking difference in affinities; cross-linking was 11-fold more efficient to the p49 subunit. The interaction of the two primase subunits with Polymerase Alpha was also investigated. Immunoprecipitation experiments indicate that only the p58 subunit directly contacts the p180 subunit of DNA Polymerase Alpha. Competition experiments in the coupled primase-Polymerase assay with a catalytically inactive mutant of DNA Polymerase Alpha and the Klenow fragment suggest that the DNA Polymerase Alpha-primase complex does not dissociate from the primer during the transition from RNA to DNA synthesis.

  • mutational studies of human DNA Polymerase Alpha identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis
    Journal of Biological Chemistry, 1993
    Co-Authors: Qun Dong, William C Copeland, Teresa S.-f. Wang
    Abstract:

    Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA Polymerase Alpha catalytic subunit. These mutants were expressed in the baculovirus system and purified to near homogeneity. The mutants had Polymerase activity ranging from 4 to 60% compared with the wild type Polymerase Alpha. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D860N, Y865S, and Y865F demonstrated no significant difference in their Km values for primer-template compared with that of the wild type enzyme. In contrast, mutants D860A, Y865S, and Y865F showed a 5-10-fold increase in the Km for deoxynucleotide triphosphate (dNTP) compared with the wild type enzyme. DNA synthetic fidelity studies of these mutants showed that mutant Y865S but not Y865F had a greater than 10-fold higher misinsertion efficiency than the wild type enzyme in Mg(2+)-catalyzed reactions. However, with Mn2+ as the metal activator, Y865S and Y865F demonstrated a 2- and 9-fold higher misinsertion efficiency, respectively. These results indicate that Asp860 and Tyr865 in region II of human DNA Polymerase Alpha are involved in incoming dNTP substrate binding. Using three deoxynucleotide structural analogs as probes, we show that the nucleotide base is the structural requirement for dNTP binding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the enzyme resistant to aphidicolin. Results of these studies strongly suggest that the phenyl ring of Tyr865 directly interacts with the nucleotide base moiety of the dNTP and plays a critical role in the misinsertion fidelity of DNA synthesis. Although mutation of Gly841 to Ala did not affect the binding of primer-template, it had a significant decrease in kcat, an increase in Km for dNTP, a striking decrease of processivity, and also resistance to aphidicolin. Thus, mutation of this residue, Gly841, which is highly conserved among the Alpha-like DNA Polymerases, appears to affect both catalysis and substrate deoxynucleotide binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the Polymerase Alpha catalytic site.

  • mutational studies of human DNA Polymerase Alpha serine 867 in the second most conserved region among Alpha like DNA Polymerases is involved in primer binding and mispair primer extension
    Journal of Biological Chemistry, 1993
    Co-Authors: Qun Dong, William C Copeland, Teresa S.-f. Wang
    Abstract:

    The second most conserved region of Alpha-like DNA Polymerases, region II, spans a block of 40 amino acid residues centered at the core sequence -DFNSLYPSII-. In the previous paper, we described mutational studies of 3 amino acid residues in region II which includes 2 amino acid residues in the core sequence. We showed that residues Asp860 and Tyr865 in the core sequence are involved in substrate deoxynucleotide triphosphate (dNTP) binding. We further showed that the phenyl moiety of the Tyr865 side chain interacts with the incoming dNTP and is responsible for the misinsertion fidelity of the enzyme. In this report, we investigated the function of 2 serine residues, Ser863 and Ser867, in this core sequence. Mutation of these 2 Ser residues to either Ala or Thr yielded mutant enzymes with similar Km for dNTPs, kcat, processivity, and misinsertion fidelity of DNA synthesis as the wild type enzyme. However, mutation of Ser867 to Ala demonstrated a 30-fold increase in Km for primer-template and a 5-fold higher KD for binding primer-template. DNA footprinting experiments of primer with the dideoxynucleotide terminus indicated that the structural feature of the primer recognized by Ser867 is the 3'-OH terminus. Single-stranded DNA inhibition data suggest that removal of the hydroxyl side chain of Ser867 affects the Polymerase's interaction with primer and not with template. Mutation of Ser867 to Ala also decreases the mutant enzyme's Km for dNTP to extend a mispaired primer and thus enhances its capacity to extend a mispaired primer terminus. These data support the conclusion that the hydroxyl side chain of Ser867 of human DNA Polymerase Alpha is involved in primer interaction during DNA synthesis and plays an essential role in mispair extension fidelity of DNA synthesis.

  • fidelity studies of the human DNA Polymerase Alpha the most conserved region among Alpha like DNA Polymerases is responsible for metal induced infidelity in DNA synthesis
    Journal of Biological Chemistry, 1993
    Co-Authors: William C Copeland, N K Lam, Teresa S.-f. Wang
    Abstract:

    Abstract Mutational studies in the highly conserved region I domain of the human DNA Polymerase Alpha enzyme demonstrated a change in metal cation-specific catalysis. Here, we extend the investigation to include the fidelity of DNA synthesis by these mutants, studying misinsertion, mispair extension, and the nucleotide analog utilization. The fidelity of region I mutants and wild type human DNA Polymerase Alpha enzyme were analyzed with either Mg2+ or Mn2+ as the metal activator. Despite the known mutagenic effect of Mn2+ in causing Polymerases to misinsert nucleotides and to utilize dideoxynucleotides, we have found that two region I mutants, D1002N and T1003S, which utilize Mn2+ in catalysis more effectively than Mg2+, actually have a 70- and 40-fold higher misinsertion fidelity, respectively, in Mn(2+)-catalyzed reactions than that of the wild type enzyme. The enhanced misinsertion fidelity of these two mutants in Mn(2+)-catalyzed reactions is due to Km discrimination of the incorrect nucleotide where the D1002N and T1003S had a 850- and 62-fold higher Km for insertion of incorrect than correct nucleotide, respectively. In Mg(2+)-catalyzed reactions, all of the region I mutants exhibited similar misinsertion efficiencies as the wild type Polymerase. Study of mispair extension showed that in Mn(2+)-catalyzed ractions, the wild type Polymerase Alpha enzyme readily extended mispair termini. In contrast, the two region I mutants, D1002N and T1003S, were unable to extend the mispaired termini in either Mg(2+)- or Mn(2+)-catalyzed reactions. These results suggest that the side chains of region I amino acids play an essential role in the Mn(2+)-induced infidelity during DNA synthesis by human DNA Polymerase Alpha. The effects of the metal activator on the utilization of two nucleotide analogs, 3'-azido-3'-deoxythymidine triphosphate and ddCTP, by the region I mutants were also investigated.

William C Copeland - One of the best experts on this subject based on the ideXlab platform.

  • the cellular DNA Polymerase Alpha primase is required for papillomavirus DNA replication and associates with the viral e1 helicase
    Proceedings of the National Academy of Sciences of the United States of America, 1994
    Co-Authors: Peter U Park, William C Copeland, Liu Yang, Teresa Wang, Michael R Botchan, Ian Mohr
    Abstract:

    Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA Polymerases. The role of the cellular DNA Polymerase Alpha-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of Polymerase Alpha inhibit DNA synthesis in this system. Addition of purified human Polymerase Alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 Polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA Polymerase activity. Moreover, this Polymerase activity can be neutralized by anti-Polymerase Alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus E1 proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular Polymerase Alpha-primase complex to the viral replication origin.

  • enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication
    Journal of Biological Chemistry, 1993
    Co-Authors: William C Copeland, Teresa S.-f. Wang
    Abstract:

    The enzymatic mechanism of primase was investigated using Escherichia coli and baculoviral overexpressed mouse primase subunits, p49 and p58. Neither of the singly purified primase subunits displayed primase activity alone, but the p49 subunit was able to extend a riboprimer, indicating that this subunit contains an RNA Polymerase activity. The p58 subunit cooperated with the p49 subunit in binding the initiating purine to form the initial dinucleotide. After initiation, the p49 subunit alone was sufficient to extend the growing primer, but both the rate of p49 primer extension and its stability were influenced by the p58 subunit. The Km(ATP) in primer synthesis on poly(dT) of the p49-p58 heterodimeric primase complex was 10-fold higher than the Km(ATP) of the single p49 subunit in a ribo(A) primer extension assay. In addition, labeled ATP cross-linked to both of the individually purified subunits but with a striking difference in affinities; cross-linking was 11-fold more efficient to the p49 subunit. The interaction of the two primase subunits with Polymerase Alpha was also investigated. Immunoprecipitation experiments indicate that only the p58 subunit directly contacts the p180 subunit of DNA Polymerase Alpha. Competition experiments in the coupled primase-Polymerase assay with a catalytically inactive mutant of DNA Polymerase Alpha and the Klenow fragment suggest that the DNA Polymerase Alpha-primase complex does not dissociate from the primer during the transition from RNA to DNA synthesis.

  • mutational studies of human DNA Polymerase Alpha identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis
    Journal of Biological Chemistry, 1993
    Co-Authors: Qun Dong, William C Copeland, Teresa S.-f. Wang
    Abstract:

    Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA Polymerase Alpha catalytic subunit. These mutants were expressed in the baculovirus system and purified to near homogeneity. The mutants had Polymerase activity ranging from 4 to 60% compared with the wild type Polymerase Alpha. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D860N, Y865S, and Y865F demonstrated no significant difference in their Km values for primer-template compared with that of the wild type enzyme. In contrast, mutants D860A, Y865S, and Y865F showed a 5-10-fold increase in the Km for deoxynucleotide triphosphate (dNTP) compared with the wild type enzyme. DNA synthetic fidelity studies of these mutants showed that mutant Y865S but not Y865F had a greater than 10-fold higher misinsertion efficiency than the wild type enzyme in Mg(2+)-catalyzed reactions. However, with Mn2+ as the metal activator, Y865S and Y865F demonstrated a 2- and 9-fold higher misinsertion efficiency, respectively. These results indicate that Asp860 and Tyr865 in region II of human DNA Polymerase Alpha are involved in incoming dNTP substrate binding. Using three deoxynucleotide structural analogs as probes, we show that the nucleotide base is the structural requirement for dNTP binding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the enzyme resistant to aphidicolin. Results of these studies strongly suggest that the phenyl ring of Tyr865 directly interacts with the nucleotide base moiety of the dNTP and plays a critical role in the misinsertion fidelity of DNA synthesis. Although mutation of Gly841 to Ala did not affect the binding of primer-template, it had a significant decrease in kcat, an increase in Km for dNTP, a striking decrease of processivity, and also resistance to aphidicolin. Thus, mutation of this residue, Gly841, which is highly conserved among the Alpha-like DNA Polymerases, appears to affect both catalysis and substrate deoxynucleotide binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the Polymerase Alpha catalytic site.

  • mutational studies of human DNA Polymerase Alpha serine 867 in the second most conserved region among Alpha like DNA Polymerases is involved in primer binding and mispair primer extension
    Journal of Biological Chemistry, 1993
    Co-Authors: Qun Dong, William C Copeland, Teresa S.-f. Wang
    Abstract:

    The second most conserved region of Alpha-like DNA Polymerases, region II, spans a block of 40 amino acid residues centered at the core sequence -DFNSLYPSII-. In the previous paper, we described mutational studies of 3 amino acid residues in region II which includes 2 amino acid residues in the core sequence. We showed that residues Asp860 and Tyr865 in the core sequence are involved in substrate deoxynucleotide triphosphate (dNTP) binding. We further showed that the phenyl moiety of the Tyr865 side chain interacts with the incoming dNTP and is responsible for the misinsertion fidelity of the enzyme. In this report, we investigated the function of 2 serine residues, Ser863 and Ser867, in this core sequence. Mutation of these 2 Ser residues to either Ala or Thr yielded mutant enzymes with similar Km for dNTPs, kcat, processivity, and misinsertion fidelity of DNA synthesis as the wild type enzyme. However, mutation of Ser867 to Ala demonstrated a 30-fold increase in Km for primer-template and a 5-fold higher KD for binding primer-template. DNA footprinting experiments of primer with the dideoxynucleotide terminus indicated that the structural feature of the primer recognized by Ser867 is the 3'-OH terminus. Single-stranded DNA inhibition data suggest that removal of the hydroxyl side chain of Ser867 affects the Polymerase's interaction with primer and not with template. Mutation of Ser867 to Ala also decreases the mutant enzyme's Km for dNTP to extend a mispaired primer and thus enhances its capacity to extend a mispaired primer terminus. These data support the conclusion that the hydroxyl side chain of Ser867 of human DNA Polymerase Alpha is involved in primer interaction during DNA synthesis and plays an essential role in mispair extension fidelity of DNA synthesis.

  • fidelity studies of the human DNA Polymerase Alpha the most conserved region among Alpha like DNA Polymerases is responsible for metal induced infidelity in DNA synthesis
    Journal of Biological Chemistry, 1993
    Co-Authors: William C Copeland, N K Lam, Teresa S.-f. Wang
    Abstract:

    Abstract Mutational studies in the highly conserved region I domain of the human DNA Polymerase Alpha enzyme demonstrated a change in metal cation-specific catalysis. Here, we extend the investigation to include the fidelity of DNA synthesis by these mutants, studying misinsertion, mispair extension, and the nucleotide analog utilization. The fidelity of region I mutants and wild type human DNA Polymerase Alpha enzyme were analyzed with either Mg2+ or Mn2+ as the metal activator. Despite the known mutagenic effect of Mn2+ in causing Polymerases to misinsert nucleotides and to utilize dideoxynucleotides, we have found that two region I mutants, D1002N and T1003S, which utilize Mn2+ in catalysis more effectively than Mg2+, actually have a 70- and 40-fold higher misinsertion fidelity, respectively, in Mn(2+)-catalyzed reactions than that of the wild type enzyme. The enhanced misinsertion fidelity of these two mutants in Mn(2+)-catalyzed reactions is due to Km discrimination of the incorrect nucleotide where the D1002N and T1003S had a 850- and 62-fold higher Km for insertion of incorrect than correct nucleotide, respectively. In Mg(2+)-catalyzed reactions, all of the region I mutants exhibited similar misinsertion efficiencies as the wild type Polymerase. Study of mispair extension showed that in Mn(2+)-catalyzed ractions, the wild type Polymerase Alpha enzyme readily extended mispair termini. In contrast, the two region I mutants, D1002N and T1003S, were unable to extend the mispaired termini in either Mg(2+)- or Mn(2+)-catalyzed reactions. These results suggest that the side chains of region I amino acids play an essential role in the Mn(2+)-induced infidelity during DNA synthesis by human DNA Polymerase Alpha. The effects of the metal activator on the utilization of two nucleotide analogs, 3'-azido-3'-deoxythymidine triphosphate and ddCTP, by the region I mutants were also investigated.

Heinz-peter Nasheuer - One of the best experts on this subject based on the ideXlab platform.

  • functional association of poly adp ribose Polymerase with DNA Polymerase Alpha primase complex a link between DNA strand break detection and DNA replication
    Nucleic Acids Research, 1998
    Co-Authors: Francoise Dantzer, Heinz-peter Nasheuer, Gilbert De Murcia, Josiane Menissierde Murcia, J L Vonesch
    Abstract:

    Poly(ADP-ribose) Polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA Polymerase Alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA Polymerase Alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA Polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template.

  • DNA replication in vitro by recombinant DNA-Polymerase-Alpha-primase.
    FEBS Journal, 1994
    Co-Authors: Franz Stadlbauer, Andrea Brueckner, Christoph Rehfuess, Verena Forster, Ben Y. Tseng, Friedrich Lottspeich, Christoph Eckerskorn, Heinz-peter Nasheuer
    Abstract:

    DNA-Polymerase-α-primase complex contains four subunits, p180, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA-Polymerase-α-primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co-expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNA-Polymerase-α-primase. The p180, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA-Polymerase-α-primase complexes showed both DNA primase and DNA Polymerase activities. The tetrameric recombinant DNA-Polymerase-α-primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.

  • cell cycle dependent phosphorylation of human DNA Polymerase Alpha
    Journal of Biological Chemistry, 1991
    Co-Authors: Heinz-peter Nasheuer, A Moore, A F Wahl, Teresa S.-f. Wang
    Abstract:

    The expression of DNA Polymerase Alpha, a principal chromosome replication enzyme, is constitutive during the cell cycle. We show in this report that DNA Polymerase Alpha catalytic polypeptide p180 is phosphorylated throughout the cell cycle and is hyperphosphorylated in G2/M phase. The p70 subunit is phosphorylated only in G2/M phase. This cell cycle-dependent phosphorylation is due to cell cycle-dependent kinase(s) and not to phosphatase(s). In vitro evidence indicates the involvement of p34cdc2 kinase in the mitotic phosphorylation of DNA Polymerase Alpha. Tryptic phosphopeptide maps demonstrate that peptides phosphorylated in vitro are identical to those phosphorylated in vivo. DNA Polymerase Alpha from mitotic cells is found to have lower affinity for single-stranded DNA than does Polymerase Alpha from G1/S phase cells. These results imply that the mitotic phosphorylation of Polymerase Alpha may affect its physical interaction with other replicative proteins and/or with DNA at the replication fork.

  • human DNA Polymerase Alpha gene sequences controlling expression in cycling and serum stimulated cells
    Molecular and Cellular Biology, 1991
    Co-Authors: B E Pearson, Heinz-peter Nasheuer, Teresa S.-f. Wang
    Abstract:

    We have investigated the DNA Polymerase Alpha promoter sequence requirements for the expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans the 5' end of this gene and includes 1.62 kb of sequence upstream from the translation start site was isolated. The transcription start site was mapped at 46 +/- 1 nucleotides upstream from the translation start site. The upstream sequence is GC rich and lacks a TATA sequence but has a CCAAT sequence on the opposite strand. Analysis of a set of deletion constructs in transient transfection assays demonstrated that efficient expression of the reporter in cycling cells requires 248 bp of sequence upstream from the cap site. Clustered within these 248 nucleotides are sequences similar to consensus sequences for Sp1-, Ap1-, Ap2-, and E2F-binding sites. The CCAAT sequence and the potential E2F- and Ap1-binding sites are shown to be protected from DNAse I digestion by partially purified nuclear proteins. The DNA Polymerase Alpha promoter can confer upon the reporter an appropriate, late response to serum addition. No single sequence element could be shown to confer serum inducibility. Rather, multiple sequence elements appear to mediate the full serum response.

M Yamada - One of the best experts on this subject based on the ideXlab platform.

  • immunoaffinity purified DNA Polymerase Alpha from a mouse temperature sensitive mutant tsft20 strain is heat labile
    Journal of Biological Chemistry, 1991
    Co-Authors: R Takadatakayama, F Hanaoka, M Yamada
    Abstract:

    Abstract We have purified DNA Polymerase Alpha from a temperature-sensitive mutant cell line of mouse FM3A cells, tsFT20, that shows temperature-sensitive activity of DNA Polymerase Alpha (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1761-1765). The purified enzyme was composed of two polypeptides with the same apparent molecular weights as those of purified DNA Polymerase Alpha from the parental strain, FM3A (Mr 180,000 and 68,000). Heat inactivation experiments revealed that this purified DNA Polymerase Alpha from tsFT20 cells was more heat-labile than the wild-type enzyme. We have also purified primase from both ts-FT20 cells and wild-type cells. Both primase fractions consist of two polypeptides with molecular weights of 54,000 and 46,000. No difference was observed between the heat labilities of the primases from tsFT20 cells and wild-type cells. Comparisons of wild-type and mutant Polymerase indicated that the temperature-sensitive mutation in DNA Polymerase Alpha from tsFT20 cells affect the dCTP-binding site of the enzyme. The mutation also changed the optimum pH and the optimum KCl concentration of the enzyme.

Paolo Plevani - One of the best experts on this subject based on the ideXlab platform.

  • the DNA Polymerase Alpha primase complex multiple functions and interactions
    The Scientific World Journal, 2003
    Co-Authors: Marco Muzifalconi, Michele Giannattasio, Marco Foiani, Paolo Plevani
    Abstract:

    DNA Polymerase Alpha (pol Alpha) holds a special position among the growing family of eukaryotic DNA Polymerases. In fact, pol Alpha is associated with DNA primase to form a four subunit complex and, as a consequence, is the only enzyme able to start DNA synthesis de novo. Because of this peculiarity the major role of the DNA Polymerase Alpha-primase complex (pol-prim) is in the initiation of DNA replication at chromosomal origins and in the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. However, pol-prim seems to play additional roles in other complex cellular processes, such as the response to DNA damage, telomere maintenance, and the epigenetic control of higher order chromatin assembly.

  • cell cycle dependent phosphorylation and dephosphorylation of the yeast DNA Polymerase Alpha primase b subunit
    Molecular and Cellular Biology, 1995
    Co-Authors: Marco Foiani, Giordano Liberi, G Lucchini, Paolo Plevani
    Abstract:

    The yeast DNA Polymerase Alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA Polymerase Alpha-primase in DNA replication.

  • the b subunit of the DNA Polymerase Alpha primase complex in saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication
    Molecular and Cellular Biology, 1994
    Co-Authors: Marco Foiani, Giovanna Lucchini, Federica Marini, Daniela Gamba, Paolo Plevani
    Abstract:

    The four-subunit DNA Polymerase Alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

  • de novo synthesis of budding yeast DNA Polymerase Alpha and pol1 transcription at the g1 s boundary are not required for entrance into s phase
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Marco Muzi Falconi, Paolo Plevani, Anna Piseri, Marina Ferrari, Giovanna Lucchini, Marco Foiani
    Abstract:

    Abstract The POL1 gene, encoding DNA Polymerase Alpha (pol Alpha) in Saccharomyces cerevisiae, is transiently transcribed during the cell cycle at the G1/S phase boundary. Here we show that yeast pol Alpha is present at every stage of the cell cycle, and its level only slightly increases following the peak of POL1 transcription. POL1 mRNA synthesis driven by a GAL1 promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pol Alpha polypeptide drops below the physiological level. Moreover, Alpha-factor-arrested cells can enter S phase and divide synchronously even if POL1 transcription is abolished. These results indicate that the level of yeast pol Alpha is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase.