Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fidelity of mammalian DNA replication and replicative DNA polymerases. 175 92

The Drosophila melanogaster gene and cDNA which span the entire open reading frame for DNA polymerase alpha, were cloned, and their nucleotide sequences were determined. The gene consists of 6 exons separated by 5 short introns. The major transcription initiation site was localized 85 bp upstream from the initiation codon. The nucleotide sequence of the open reading frame revealed a polypeptide of 1,505 amino acid residues with a molecular weight of 170,796. The amino acid sequence of the polypeptide was 37% homologous with that of the catalytic subunit of human DNA polymerase alpha. This sequence contains six regions, the orders and amino acid sequences of which are highly conserved among a number of other viral and eukaryotic DNA polymerases. We found 7 amino acid residues in the region between the 639th and 758th positions, identical to those essential for the active site of Escherichia coli DNA polymerase I-associated 3'----5' exonuclease. Thus, the exonuclease activity may be associated with Drosophila DNA polymerase alpha. Levels of the DNA polymerase alpha mRNA were high in unfertilized eggs and early embryos, relatively high in adult female flies and second-instar larva, and low in bodies at other stages of development. This feature of the expression is similar to that of the proliferating cell nuclear antigen (an auxiliary protein of DNA polymerase delta) and seems to coincide with the proportions of proliferating cells in various developmental stages. As the half life of the mRNA for DNA polymerase alpha in cultured Drosophila Kc cells was 15 min, expression of the DNA polymerase alpha gene is probably strictly regulated at the step of transcription.
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PMID:Structure and expression during development of Drosophila melanogaster gene for DNA polymerase alpha. 192 67

The proliferative capacity of brain-tumor cells was analyzed in vitro and in situ using monoclonal antibody (MAb) against deoxyribonucleic acid (DNA) polymerase alpha. For the in vitro studies, two cultured human glioma cell lines were investigated using MAb against DNA polymerase alpha, the MAb Ki-67, a serum against proliferating cell nuclear antigen (PCNA/cyclin), bromodeoxyuridine (BUdR), and an anti-BUdR MAb. During exponential growth of the cells, the percentage of polymerase alpha-positive cells (the "polymerase alpha score") ranged from 72.0% to 77.1%, the Ki-67-positive cells (the "Ki-67 score") ranged from 43.4% to 59.4%, the PCNA/cyclin-positive cells from 30.9% to 41.4%, and the BUdR labeling index from 28.6% to 39.3%. For the in situ studies, tissue from 60 human brain tumors and from two normal human brains was investigated and the polymerase alpha scores and Ki-67 scores were compared. In normal brain tissue, no immunostaining was found by either method. In brain tumors, both the polymerase alpha scores and the Ki-67 scores correlated with the histological grade of malignancy. Polymerase alpha scores were generally higher than Ki-67 scores in the same specimen, especially in malignant brain tumors. These findings suggest that immunostaining of DNA polymerase alpha is a convenient and important new method by which to estimate the cellular proliferation rate of brain tumors. Polymerase alpha scores may be closer to the growth fraction of the individual tumor than the MAb Ki-67 or other scores.
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PMID:Immunohistochemical demonstration of DNA polymerase alpha in human brain-tumor cells. 196 2

The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication. It contains both DNA polymerase alpha (I) and delta (III). Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast. We have now cloned the gene for yeast PCNA (POL30). The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA. Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene. Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase. In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis.
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PMID:Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. 197 Jan 60

In the presence of a single-stranded-DNA-binding protein (SSB), the elongation of primed DNA templates by DNA polymerase delta (pol delta) is dependent on ATP and two protein factors, activator 1 (A1) and proliferating cell nuclear antigen (PCNA). We have examined the interaction of these proteins with (dA)4500.(dT)12-18 by measuring their ability to form stable complexes with this DNA. In the presence of ATP, A1, PCNA, and pol delta formed a stable complex with DNA that could be isolated by gel filtration. Incubation of the isolated complex with dTTP resulted in the synthesis of poly(dT). While ATP was required for the formation of this complex, it was not required for the subsequent elongation of DNA. The temporal requirements for complex formation were determined. A1 was found to bind first, followed by the ATP-dependent addition of PCNA to the A1.DNA complex, while pol delta was added last. Each of these complexes could be isolated by gel filtration, indicating that they possessed a high degree of stability. The binding of PCNA to the A1-SSB-coated primed DNA occurred with adenosine 5'-[gamma-thio]triphosphate as well as ATP. However, the binding of pol delta to the PCNA.A1-DNA complex was observed only when the latter complex was formed in the presence of ATP. The complete complex was formed after incubation at 37 degrees C for 2 min, whereas no complex was detected after incubation at 0 degree C. These results indicate that these proteins act in a manner analogous to the accessory proteins that play critical roles in the elongation reaction catalyzed by T4 phage DNA polymerase and Escherichia coli DNA polymerase III.
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PMID:Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1. 197 50

DNA polymerases alpha, delta, and epsilon have been purified and characterized from the same HeLa cell extract in order to determine their relationship by comparing them from the same cell type. The catalytic properties and the primary structures of the large subunits of the DNA polymerases as compared by partial peptide mapping with N-chlorosuccinimide are different. Likewise, the small subunit of DNA polymerase epsilon appears to be distinct from the large subunit of the same polymerase and from the smaller subunits of DNA polymerase alpha. HeLa DNA polymerase delta is processive only when HeLa proliferating cell nuclear antigen is present, whereas DNA polymerase epsilon is quite processive in its absence. Inhibitor and activator spectra of DNA polymerases alpha, delta, and epsilon also distinguish the three enzymes. These results and immunologic comparisons published elsewhere support the premise that HeLa DNA polymerases alpha, delta, and epsilon are distinct enzymes that have common properties with yeast DNA polymerases I, III, and II, respectively.
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PMID:DNA polymerases alpha, delta, and epsilon: three distinct enzymes from HeLa cells. 197 94

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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PMID:Two forms of DNA polymerase delta from mouse cells. Purification and properties. 197 12

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.
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PMID:Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon. 200 86

The catalytic core protomer of calf thymus DNA polymerase delta (pol delta) was purified to apparent homogeneity by a modified procedure, and its enzymologic mechanism was investigated using a combination of steady-state kinetics and semiquantitative sedimentation binding analyses. Like DNA polymerase alpha (pol alpha), in the absence of a primer, pol delta was able to bind single-stranded but not double-stranded DNA. This, in conjunction with the observation of induced substrate (dNTP) inhibition of pol delta in the presence of a correctly base-paired 2',3'-dideoxyribonucleotide-terminated primer, suggests that pol delta follows an ordered sequential ter-reactant mechanism of substrate recognition and binding similar to that elucidated for pol alpha. Pol delta binds template first followed by primer and then template-directed dNTP. With suitable substrates, addition to incubations of proliferating cell nuclear antigen, the pol delta auxiliary factor, leads to a reduction in Km and increase in Vmax. This suggests that proliferating cell nuclear antigen enhances the processivity of pol delta by increasing both the residence time of pol delta on the DNA template-primer and the rate at which individual nucleotides are incorporated.
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PMID:Enzymologic mechanism of calf thymus DNA polymerase delta. 205 Jun 71

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.
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PMID:DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases. 208 20


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