The Experts below are selected from a list of 198 Experts worldwide ranked by ideXlab platform
Ulrich Hubscher - One of the best experts on this subject based on the ideXlab platform.
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DNA Polymerase switching ii replication factor c abrogates primer synthesis by DNA Polymerase alpha at a critical length
Journal of Molecular Biology, 2000Co-Authors: Romina Mossi, Robert Keller, Elena Ferrari, Ulrich HubscherAbstract:Abstract A crucial event in DNA replication is the Polymerase switch from the synthesis of a short RNA/DNA primer by DNA Polymerase α/primase to the pro?cessive elongation by DNA Polymerase δ. In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA Polymerase α were investigated. We show that RF-C stalls DNA Polymerase α after synthesis of approximately 30 nucleotides, while not inhibiting the Polymerase activity per se . This suggested that RF-C and the length of the primer may be two important factors contributing to the Polymerase switch. Furthermore the DNA binding properties of RF-C were tested. Band shift experiments indicated that RF-C has a preference for 5′ recessed ends and double-stranded DNA over 3′ ends. Finally PCNA can be loaded onto a DNA template carrying a RNA primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp. Thus we propose a model where RF-C, upon binding to the RNA/DNA primer, influences primer synthesis and sets the conditions for a Polymerase switch after recruiting PCNA to DNA.
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DNA replication machinery functional characterization of a complex containing DNA Polymerase alpha DNA Polymerase delta and replication factor c suggests an asymmetric DNA Polymerase dimer
Biochemistry, 1996Co-Authors: Giovanni Maga, Ulrich HubscherAbstract:By using a complementation assay for a replication factor C dependent DNA Polymerase activity on a singly-primed M13 DNA template, we have isolated from calf thymus a multiprotein complex active in DNA replication. For this, the inclusion of ATP during the entire isolation procedure was essential, since the complex decayed after omission of ATP. This complex contains at least DNA Polymerase α/primase, DNA Polymerase δ, and replication factor C as shown by gel-filtration and coimmunoprecipitation experiments. It is functionally active in replication of primed and unprimed single-stranded M13 DNA templates. Furthermore, in the presence of proliferating cell nuclear antigen and ATP, it forms an isolatable holoenzyme/template-primer complex. Replication factor C apparently mediates the interaction of DNA Polymerase δ in the complex with proliferating cell nuclear antigen, through an ATP-dependent mechanism. This interaction appears to stabilize the binding of the complex to a template-primer and to coordinate...
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DNA Polymerase ε: in search of a function
Trends in Biochemical Sciences, 1992Co-Authors: Ulrich Hubscher, Pia ThömmesAbstract:Abstract The current model of eukaryotic DNA replication involves the two DNA Polymerases δ and α as the leading and lagging strand enzymes, respectively. A DNA Polymerase first discovered in yeast has now been found in all eukaryotic cells and is termed DNA Polymerase e. In yeast, the gene for DNA Polymerase e has recently been found to be essential for viability, raising new questions about its functions.
N. Lan Toomey - One of the best experts on this subject based on the ideXlab platform.
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characterization of human DNA Polymerase delta and its immunochemical relationships with DNA Polymerase alpha and epsilon
Journal of Biological Chemistry, 1991Co-Authors: Yunquan Jiang, Shan Jian Zhang, N. Lan ToomeyAbstract: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.
Giuseppe Baldacci - One of the best experts on this subject based on the ideXlab platform.
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inter species DNA Polymerase delta chimeras are functional in saccharomyces cerevisiae
FEBS Journal, 1995Co-Authors: Genevieve Moussy, Anne-marie De Recondo, Giuseppe BaldacciAbstract:The catalytic subunits of DNA Polymerase δ of Schizosaccharomyces pombe and Saccharomyces cerevisiae share over 50% identity. The capability of S. pombe DNA Polymerase δ to complement two thermosensitive mutants of S. cerevisiae was studied in vivo and it was determined that complementation was allele dependent. However, DNA Polymerase δ from S. pombe did not restore growth of a S. cerevisiae strain containing a disrupted chromosomal copy of the POL3 gene that encodes DNA Polymerase δ. To identify the regions of DNA Polymerase δ responsible for species-specific interactions, we constructed different chimeras with S. cerevisiae and S. pombe DNA Polymerase δ genes. The growth of a S. cerevisiae strain with a disrupted chromosomal POL3 gene was studied after transformation with plasmids expressing different chimeras. A 1254-bp region located in the 3′ region of the S. cerevisiae POL3 gene is responsible for species-specific functions.
Susan Grimm - One of the best experts on this subject based on the ideXlab platform.
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two forms of DNA Polymerase delta from mouse cells purification and properties
Journal of Biological Chemistry, 1990Co-Authors: Mehran Goulian, S M Herrmann, J W Sackett, Susan GrimmAbstract:Abstract A procedure is described for the purification from cultured mouse cells of two DNA Polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA Polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA Polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA Polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA Polymerase delta, and multiple similarities with DNA Polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA Polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA Polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA Polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA Polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA Polymerase delta-like enzymes with higher molecular mass than the Polymerase delta of Lee et al., which have recently been named DNA Polymerases epsilon).
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Purification and properties of an accessory protein for DNA Polymerase alpha/primase.
Journal of Biological Chemistry, 1990Co-Authors: Mehran Goulian, Cheryl J. Heard, Susan GrimmAbstract:: A protein that stimulates DNA Polymerase alpha/primase many-fold on unprimed poly(dT) was purified to homogeneity from extracts of cultured mouse cells. The protein contains polypeptides of approximately 132 and 44 kDa, and the total molecular mass of 150 kDa calculated from Stokes radius (54 A) and sedimentation coefficient (6.7 S) indicates that it contains one each of the two subunits. The purified "alpha accessory factor" (AAF) also stimulates DNA Polymerase alpha/primase in the self-primed reaction with unprimed single-stranded DNA. In addition to these effects on the coordinate activities of DNA Polymerase alpha and DNA primase, stimulatory effects were also demonstrated separately on both the Polymerase and primase activities of the enzyme complex. However, there was no stimulation with DNAse-treated ("activated") DNA under normal conditions for assay of DNA Polymerase alpha. The stimulatory activity of mouse AAF is highly specific for DNA Polymerase alpha/primase; no effect was observed with mouse DNA Polymerases beta, gamma, or delta, nor with retroviral, bacteriophage, or bacterial DNA Polymerases. Mouse AAF stimulated human DNA Polymerase alpha/primase with several different templates, similar to results with the mouse enzyme. However, it had very little effect on the DNA Polymerase/primase from either Drosophila embryo or from yeast.
Michel Castroviejo - One of the best experts on this subject based on the ideXlab platform.
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Wheat DNA Polymerase CI: a homologue of rat DNA Polymerase beta.
Plant Molecular Biology, 1998Co-Authors: A.e. Luque, J.p. Benedetto, Michel CastroviejoAbstract:We have previously described a low-molecular-weight DNA Polymerase (52 kDa) from wheat embryo: DNA Polymerase CI (pol CI). This enzyme shares some biochemical properties with animal DNA Polymerase β (pol β). In this report, we analyse pol CI in wheat embryo germination. Immunodetection and measurement of the enzyme activity show that wheat pol CI remains at a constant level during germination, whereas dramatic changes of the replicative DNA Polymerase A and B activities were previously reported. We observe that the level of pol CI in physiological conditions (embryo germination and dividing cell culture) is in agreement with a pol β-type DNA Polymerase. By microsequencing of the electroblotted 52 kDa polypeptide, we determined the sequence of a dodecapeptide from the N-terminal region. A comparative analysis of the N-terminal pol CI peptide with some mammalian pol β sequences shows a clear homology with helix 1 of the N-terminal ssDNA domain (residues 15 to 26) of the rat pol β. Thus, the helical structure of this region should be conserved in the wheat peptide. This represents the first evidence of a partial primary structure of a β-type DNA Polymerase in plants.
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DNA Polymerase B from wheat embryos: A plant δ-like DNA Polymerase
Archives of Biochemistry and Biophysics, 1991Co-Authors: Marie Claude Richard, Simon Litvak, Michel CastroviejoAbstract:Abstract Studies in eucaryotic cells (mainly animals and yeast) indicate that at least two DNA Polymerases are involved in DNA replication at the level of the replication fork: DNA Polymerase α, which is associated with DNA primase, is involved in the replication of the lagging strand; DNA Polymerase δ, associated with an exonuclease activity, synthesizes the forward continuous DNA strand. Much less information exists concerning plant systems. Previous work from this laboratory provided preliminary evidence of an association between DNA Polymerase B from wheat embryo and an exonucleolytic activity. In this paper, we present additional data on the biochemical properties of DNA Polymerase B. An improved purification procedure described in this article has been developed. During all the purification steps the nuclease activity was associated with DNA Polymerase activity. A biochemical study of this enzyme activity shows that it is an exonuclease which hydrolyses DNA in the 3′ to 5′ direction. Moreover, this exonuclease confers a proofreading function to DNA Polymerase B. Comparison of DNA Polymerase B properties (template specificity, sensitivity to DNA replication inhibitors like aphidicolin and butyl-phenyl dGTP, copurification of DNA Polymerase and exonuclease activities) with those of animal DNA Polymerase δ indicates that these enzymes share many common features. To our knowledge, this is the first report of DNA Polymerase δ in higher plants.
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A low molecular weight DNA Polymerase from wheat embryos
Plant Molecular Biology, 1990Co-Authors: Michel Castroviejo, Marie-thérèse Gatius, Simon LitvakAbstract:The study of plant DNA Polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA Polymerase (DNA Polymerase C_I) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA Polymerase C_I did not inhibit animal DNA Polymerases α and β or wheat DNA Polymerase A, whereas wheat DNA Polymerases C_II and B were much less affected than the C_I enzyme. Several properties of enzyme C_I were studied. Some known inhibitors of DNA Polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA Polymerase C_I while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although Polymerase C_I can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA Polymerase β. The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal Polymerase) indicate that the DNA Polymerase described in this work is a plant DNA Polymerase very similar to the low molecular weight animal DNA Polymerase β, an enzyme believed to be involved in nuclear DNA repair.