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

  • identification of Delta helicase as the bovine homolog of hupf1 demonstration of an interaction with the third subunit of DNA Polymerase Delta
    Nucleic Acids Research, 2002
    Co-Authors: Michael L Carastro, A G So, Manuel Selg, Hansmartin Jack, K M Downey
    Abstract:

    Delta helicase is a5 ′ to 3′ DNA helicase that partially co-purifies with DNA Polymerase Delta (pol Delta) from fetal bovine thymus tissue. We describe the resolution of Delta helicase from pol Delta on heparin–agarose chromatography and its purification to apparent homogeneity by affinity purification on single-stranded DNA–cellulose chromatography, unique-sequence RNA–agarose chromatography, and ceramic hydroxyapatite chromatography. Delta helicase isolated from fetal bovine thymus had an apparent M r of 115 kDa in SDS–PAGE, and photocrosslinked to [α-32P]ATP. Tandem mass spectrometry peptide mass data derived from the bovine polypeptide matched to human UPF1 (HUPF1), a 5′ to 3′ RNA and DNA helicase, and a requisite component of the mRNA surveillance complex. Antisera against HUPF1 cross-reacted with Delta helicase on western analysis, and Delta helicase activity was immunoinactivated by pre-incubation with antibodies to HUPF1, suggesting that Delta helicase is the bovine homolog of HUPF1. Immunoprecipitation experiments demonstrated that HUPF1 interacts with the 66-kDa third subunit of pol Delta in vivo.

  • The small subunit is required for functional interaction of DNA Polymerase δ with the proliferating cell nuclear antigen
    Nucleic Acids Research, 1997
    Co-Authors: Jin-qiu Zhou, K M Downey, Hua He, A G So
    Abstract:

    : DNA Polymerase Delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA Polymerase Delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA Polymerase Delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA Polymerase Delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA Polymerase Delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA Polymerase Delta with PCNA and for highly processive DNA synthesis.

  • schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA Polymerase Delta processivity
    Journal of Biological Chemistry, 1996
    Co-Authors: Martha P Arroyo, A G So, K M Downey, Teresa S F Wang
    Abstract:

    Abstract We introduced nine site-directed mutations into seven conserved fission yeast proliferative cell nuclear antigen (PCNA) residues, Leu2, Asp63, Arg64, Gly69, Gln201, Glu259, and Glu260, either as single or as double mutants. Both the recombinant wild type and mutant PCNAs were able to form homotrimers in solution and to sustain growth of a null pcna strain (Δpcna). Wild type Schizosaccharomyces pombe PCNA and PCNA proteins with mutations in Asp63, Gln201, Glu259, or Glu260 to Ala were able to stimulate DNA synthetic activity and to enhance the processivity of calf thymus DNA Polymerase δ holoenzyme similar to calf thymus PCNA. Mutations of Leu2 to Val or Arg64 to Ala, either singly or as a double mutant, yielded PCNA mutant proteins that had reduced capacity in enhancing the processivity of DNA Polymerase δ but showed no deficiency in stimulation of the ATPase activity of replication factor C. S. pombe Δpcna strains sustained by these two mutant-pcna alleles had moderate defects in growth and displayed elongated phenotypes. These cells, however, were not sensitive to UV irradiation. Together, these in vitro and in vivo studies suggest that the side chains of Leu2 and Arg64 in one face of the PCNA trimer ring structure are two of the several sites involved in tethering DNA Polymerase δ for processive DNA synthesis during DNA replication.

  • the mammalian DNA Polymerase Delta proliferating cell nuclear antigen template primer complex molecular characterization by direct binding
    Biochemistry, 1996
    Co-Authors: Maeve Mcconnell, K M Downey, Holly Miller, Dmitry Ju Mozzherin, Aaron Quamina, Paul A Fisher
    Abstract:

    : Three direct assays, polyacrylamide gel electrophoresis-band mobility shift, agarose gel electrophoresis-band mobility shift, and nitrocellulose filter binding, were established to study complexes formed among mammalian DNA Polymerase Delta (pol Delta), proliferating cell nuclear antigen (PCNA), and synthetic oligonucleotide template-primers. In all contexts, complex formation requires simultaneous presence of pol Delta, PCNA, and template-primer. Moreover, we showed in one such assay that the complex formed contains each molecular component. Nuclease protection experiments demonstrate that complex formation protects template from degradation by DNAse I. The mass determined for the pol Delta.PCNA.template-primer complex was about 267 kDa, consistent with the participation of one molecule of pol Delta, two or three molecules of PCNA and one molecule of template-primer. PCNA alone behaved as a trimer (mass determined to be about 87 kDa). Complex could be manipulated enzymologically. Measurement of off rates demonstrates directly that PCNA stabilizes the pol Delta.template-primer complex.

  • interaction of DNA Polymerase Delta proliferating cell nuclear antigen and synthetic oligonucleotide template primers analysis by polyacrylamide gel electrophoresis band mobility shift assay
    Journal of Biological Chemistry, 1993
    Co-Authors: L Ng, K M Downey, Maeve Mcconnell, Paul A Fisher
    Abstract:

    Abstract A polyacrylamide gel electrophoresis band-mobility shift assay was developed to study the binding of synthetic oligonucleotides by DNA Polymerase Delta (pol Delta) and proliferating cell nuclear antigen (PCNA). As measured by this assay, neither calf thymus pol Delta core enzyme nor PCNA alone bind DNA stably. However, mammalian PCNA but not Drosophila PCNA promotes the formation of a distinct pol Delta.PCNA.template-primer complex. Appearance of this complex is primer-dependent but does not require Mg2+. Complex stability is also influenced by the presence or absence of individual dNTPs. A model for the ordered sequential interaction of pol Delta, PCNA, and DNA template-primers is proposed.

A G So - One of the best experts on this subject based on the ideXlab platform.

  • identification of Delta helicase as the bovine homolog of hupf1 demonstration of an interaction with the third subunit of DNA Polymerase Delta
    Nucleic Acids Research, 2002
    Co-Authors: Michael L Carastro, A G So, Manuel Selg, Hansmartin Jack, K M Downey
    Abstract:

    Delta helicase is a5 ′ to 3′ DNA helicase that partially co-purifies with DNA Polymerase Delta (pol Delta) from fetal bovine thymus tissue. We describe the resolution of Delta helicase from pol Delta on heparin–agarose chromatography and its purification to apparent homogeneity by affinity purification on single-stranded DNA–cellulose chromatography, unique-sequence RNA–agarose chromatography, and ceramic hydroxyapatite chromatography. Delta helicase isolated from fetal bovine thymus had an apparent M r of 115 kDa in SDS–PAGE, and photocrosslinked to [α-32P]ATP. Tandem mass spectrometry peptide mass data derived from the bovine polypeptide matched to human UPF1 (HUPF1), a 5′ to 3′ RNA and DNA helicase, and a requisite component of the mRNA surveillance complex. Antisera against HUPF1 cross-reacted with Delta helicase on western analysis, and Delta helicase activity was immunoinactivated by pre-incubation with antibodies to HUPF1, suggesting that Delta helicase is the bovine homolog of HUPF1. Immunoprecipitation experiments demonstrated that HUPF1 interacts with the 66-kDa third subunit of pol Delta in vivo.

  • The small subunit is required for functional interaction of DNA Polymerase δ with the proliferating cell nuclear antigen
    Nucleic Acids Research, 1997
    Co-Authors: Jin-qiu Zhou, K M Downey, Hua He, A G So
    Abstract:

    : DNA Polymerase Delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA Polymerase Delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA Polymerase Delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA Polymerase Delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA Polymerase Delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA Polymerase Delta with PCNA and for highly processive DNA synthesis.

  • schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA Polymerase Delta processivity
    Journal of Biological Chemistry, 1996
    Co-Authors: Martha P Arroyo, A G So, K M Downey, Teresa S F Wang
    Abstract:

    Abstract We introduced nine site-directed mutations into seven conserved fission yeast proliferative cell nuclear antigen (PCNA) residues, Leu2, Asp63, Arg64, Gly69, Gln201, Glu259, and Glu260, either as single or as double mutants. Both the recombinant wild type and mutant PCNAs were able to form homotrimers in solution and to sustain growth of a null pcna strain (Δpcna). Wild type Schizosaccharomyces pombe PCNA and PCNA proteins with mutations in Asp63, Gln201, Glu259, or Glu260 to Ala were able to stimulate DNA synthetic activity and to enhance the processivity of calf thymus DNA Polymerase δ holoenzyme similar to calf thymus PCNA. Mutations of Leu2 to Val or Arg64 to Ala, either singly or as a double mutant, yielded PCNA mutant proteins that had reduced capacity in enhancing the processivity of DNA Polymerase δ but showed no deficiency in stimulation of the ATPase activity of replication factor C. S. pombe Δpcna strains sustained by these two mutant-pcna alleles had moderate defects in growth and displayed elongated phenotypes. These cells, however, were not sensitive to UV irradiation. Together, these in vitro and in vivo studies suggest that the side chains of Leu2 and Arg64 in one face of the PCNA trimer ring structure are two of the several sites involved in tethering DNA Polymerase δ for processive DNA synthesis during DNA replication.

  • primary structure of the catalytic subunit of calf thymus DNA Polymerase Delta sequence similarities with other DNA Polymerases
    Biochemistry, 1991
    Co-Authors: Jian Zhang, Dominic W Chung, K M Downey, Earl W Davie, A G So
    Abstract:

    : The 125- and 48-kDa subunits of bovine DNA Polymerase Delta have been isolated by SDS-polyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA Polymerase Delta preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116-kDa polypeptides were used to design primers for the Polymerase chain reaction to determine the nucleotide sequence of a full-length cDNA encoding the catalytic subunit of bovine DNA Polymerase Delta. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123,707. This is in agreement with the molecular weight of 125,000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA Polymerase Delta with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA Polymerase Delta contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA Polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA Polymerase Delta, but these regions share little or no homology with the alpha Polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA Polymerases, whereas one region is not homologous to any other DNA Polymerase that has been sequenced thus far.(ABSTRACT TRUNCATED AT 250 WORDS)

  • primary structure of the catalytic subunit of human DNA Polymerase Delta and chromosomal location of the gene
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Dominic W Chung, A G So, Jian Zhang, Earl W Davie, K M Downey
    Abstract:

    Abstract The catalytic subunit (Mr approximately 124,000) of human DNA Polymerase Delta has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA Polymerase Delta. The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids. The enzyme was 94% identical to bovine DNA Polymerase Delta and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA Polymerase Delta was localized to chromosome 19.

Ulrich Hubscher - One of the best experts on this subject based on the ideXlab platform.

  • mediation of proliferating cell nuclear antigen pcna dependent DNA replication through a conserved p21 cip1 like pcna binding motif present in the third subunit of human DNA Polymerase Delta
    Journal of Biological Chemistry, 2001
    Co-Authors: Manuelle Ducoux, Serge Urbach, Giuseppe Baldacci, Ulrich Hubscher, Stephane Koundrioukoff, Jesper Christensen, Patrick Hughes
    Abstract:

    The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA Polymerase Delta has not been clearly defined. We show that the third subunit of human DNA Polymerase Delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA Polymerase Delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA Polymerase Delta. Furthermore, pull-down assays showed that DNA Polymerase Delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA Polymerase Delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA Polymerase Delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA Polymerase Delta.

  • the pcna from thermococcus fumicolans functionally interacts with DNA Polymerase Delta
    Biochemical and Biophysical Research Communications, 2000
    Co-Authors: Ghislaine Henneke, Elena Ferrari, Jeanpaul Raffin, Zophonias O Jonsson, Jacques Dietrich, Ulrich Hubscher
    Abstract:

    We have cloned the gene encoding proliferating cell nuclear antigen (PCNA) from the hyperthermophilic euryarchaeote Thermococcus fumicolans (Tfu). Tfu PCNA contains 250 amino acids with a calculated Mr of 28,000 and is 26% identical to human PCNA. Next, Tfu PCNA was overexpressed in Escherichia coli and it showed an apparent molecular mass of 33.5 kDa. The purified Tfu PCNA was tested first with recombinant Tfu DNA Polymerase I (Tfu pol) and second with calf thymus DNA Polymerase δ (pol δ). When tested with the homologous Tfu pol on bacteriophage λ DNA, large amounts of Tfu PCNA were required to obtain two- to threefold stimulation. Surprisingly, however, Tfu PCNA was much more efficient than human PCNA in stimulating calf thymus pol δ. Our data suggest that PCNA has been functionally conserved not only within eukaryotes but also from hyperthermophilic euryarchaeotes to mammals.

  • mutant DNA Polymerase Delta from thermosensitive schizosaccharomyces pombe strains display reduced stimulation by proliferating cell nuclear antigen
    Biochemical Journal, 1998
    Co-Authors: Mylene Perderiset, Ulrich Hubscher, Giovanni Maga, Karine Piard, Stefania Francesconi, Isabelle Tratner, Giuseppe Baldacci
    Abstract:

    : We have isolated and characterized DNA Polymerase Delta (pol Delta) from two thermosensitive Schizosaccharomyces pombe strains, polDeltats1 and polDeltats3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 37 degreesC polDeltats1 and polDeltats3 mutant strains arrest growth in the S phase of the cell cycle. We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol Delta. Affinity for primer (3'-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol Delta. In contrast, Vmax values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis. In addition, ts1 pol Delta is not stimulated by low PCNA concentration at 37 degreesC, although low concentrations stimulate activity at 25 degreesC, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol Delta mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.

  • DNA Polymerase Delta an essential enzyme for DNA transactions
    Biological Chemistry, 1997
    Co-Authors: Robert Hindges, Ulrich Hubscher
    Abstract:

    : Many DNA transactions, such as replication, repair and recombination involve DNA synthesis and consequently require the action of DNA synthesizing enzymes called DNA Polymerases (Pol). Eukaryotic cells contain at least six different Pols, named alpha, beta, gamma, Delta, epsilon, and zeta. Among them Pol Delta occupies important roles in DNA replication, nucleotide excision repair, base excision repair and VDJ recombination. Pol a has been extremely conserved in evolution from yeast to man. The function of Pol Delta must be considered in the context of two other factors, called proliferating cell nuclear antigen and replication factor C, two protein complexes that build together the moving platform for Pol Delta. This moving platform provides an important framework for dynamic properties of an accurate Pol Delta such as its recruitment when its function is needed, the facilitation of Pol Delta binding to the primer terminus, the increase in Pol Delta processivity, the prevention of non-productive binding of the Pol Delta to single-stranded DNA, the release of Pol Delta after DNA synthesis and the bridging of Pol Delta interactions to other replication proteins. In this review we summarize the current knowledge of Pol Delta and will focus in particular to its structural conservation, its functional tasks in the cell and its interactions with other proteins.

  • regulation of DNA metabolic enzymes upon induction of preb cell development and v d j recombination up regulation of DNA Polymerase Delta
    Nucleic Acids Research, 1997
    Co-Authors: Rolf Jessberger, Primo Schar, Peter Robins, Elena Ferrari, Brigitte Riwar, Ulrich Hubscher
    Abstract:

    Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA Polymerases alpha, beta, Delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA Polymerase Delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA Polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA Polymerase Delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.

John J Byrnes - One of the best experts on this subject based on the ideXlab platform.

  • pcna dependent DNA Polymerase Delta from rabbit bone marrow
    Biochemistry, 1992
    Co-Authors: Changde Lu, John J Byrnes
    Abstract:

    : Proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA Polymerase Delta were partially purified and characterized from rabbit bone marrow. Rabbit DNA Polymerase Delta sediments at 8.2 S upon glycerol density gradient centrifugation. Similar to calf thymus PCNA-dependent DNA Polymerase Delta, a 125-123-kDa doublet and 48-kDa polypeptides correlate with DNA Polymerase activity. Western blotting of rabbit DNA Polymerase Delta with polyclonal antibody to calf thymus PCNA-dependent DNA Polymerase Delta gives the same results as calf thymus Delta; the 125-123-kDa doublet is recognized. PCNA-dependent DNA Polymerase Delta is resistant to inhibition by dideoxynucleotides and is relatively insensitive to inhibition by N2-[p-(n-butyl)phenyl]dGTP. A 3'-->5' exonuclease copurifies with the DNA Polymerase. The processivity of DNA Polymerase Delta alone is very low but greatly increases with the addition of PCNA from rabbit bone marrow or calf thymus. Comparative studies of the original DNA Polymerase Delta from rabbit bone marrow demonstrate a lack of recognition by antibodies to calf thymus Delta and a high degree of processivity in the absence of PCNA. Additionally, the originally described DNA Polymerase Delta is a single polypeptide of 122 kDa. These features would recategorize the original Delta to the epsilon category by recently proposed convention. PCNA-dependent DNA Polymerase Delta is a relatively minor component of rabbit bone marrow compared to DNA Polymerase alpha and PCNA-independent DNA Polymerase Delta (epsilon), the relative proportions being alpha, 60%; Delta, 7%; and epsilon, 30%.

Shan Jian Zhang - One of the best experts on this subject based on the ideXlab platform.

  • DNA Polymerase Delta is involved in the cellular response to uv damage in human cells
    Journal of Biological Chemistry, 1994
    Co-Authors: Xiaorong Zeng, Yunquan Jiang, Shan Jian Zhang
    Abstract:

    Abstract We have used antibodies specific for either Polymerase Delta (pol Delta) or its accessory protein, proliferating cell nuclear antigen (PCNA), to demonstrate that they can markedly inhibit the capacity of HeLa nuclear extracts to effect repair of UV-damaged plasmid DNA. This provides the first unambiguous evidence for the involvement of pol Delta in DNA repair synthesis. The mRNA levels of both pol Delta and PCNA were significantly stimulated subsequent to UV irradiation of cultured cells, providing the first evidence that the cellular response to UV damage may involve regulation of pol Delta and PCNA at the gene level. Thus, DNA Polymerase Delta and its accessory proteins, in addition to their function in replicative DNA synthesis, also function in DNA repair synthesis.

  • Structural and functional relationships of human DNA Polymerases
    Chromosoma, 1992
    Co-Authors: Yunquan Jiang, Shan Jian Zhang, P Zhang, R X Zeng
    Abstract:

    A continuing theme of our laboraory, has been the understanding of human DNA Polymerases at the structural level. We have purified DNA Polymerases Delta, epsilon and alpha from human placenta. Monoclonal antibodies to these Polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA Polymerases Delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA Polymerase Delta and epsilon. We have cloned the DNA Polymerase Delta cDNA, and this has allowed us to compare its primary structure to those of human Polymerase alpha and other members of this Polymerase family. Multiple sequence alignments have revealed that human DNA Polymerase Delta is also closely related to the herpes virus family of DNA Polymerases. In situ hybridization has shown that the human DNA Polymerase Delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA Polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA Polymerase δ during the cell cycle.

  • characterization of human DNA Polymerase Delta and its immunochemical relationships with DNA Polymerase alpha and epsilon
    Journal of Biological Chemistry, 1991
    Co-Authors: Yunquan Jiang, Shan Jian Zhang, N L Toomey
    Abstract:

    Abstract DNA Polymerase Delta was purified from human placenta and its Polymerase catalytic subunit identified as a 125-kDa polypeptide by activity staining. This 125-kDa form of DNA Polymerase Delta resembles that reported from calf thymus (Lee, M. Y. W. T., Tan, C.-K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913) and differs in molecular properties from a previously described form isolated from human placenta (Lee, M. Y. W. T., and Toomey, N. L. (1987) Biochemistry 26, 1076-1085) and now referred to as DNA Polymerase epsilon. The properties of DNA Polymerase Delta were further investigated to determine its relationships with DNA Polymerase epsilon. The two enzymes differed in their response to proliferating cell nuclear antigen. Monoclonal antibodies against DNA Polymerase Delta were raised and used to examine its immunochemical relationships with DNA Polymerase alpha and epsilon. These studies provided evidence that all three proteins are structurally distinct but share a common epitope(s). Immunofluorescence microscopy indicates that DNA Polymerase Delta and possibly also DNA Polymerase epsilon are localized to the nucleus.