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)

Immunohistochemical detection of cell cycle-related markers for estimation of tumor growth fractions using paraffin-embedded tissue sections would have applications in experimental and clinical pathology as an in situ histologic alternative to flow cytometry. The monoclonal antibodies 19A2 and PC10 detect the proliferating cell nuclear antigen (PCNA/Cyclin), an auxiliary protein to DNA polymerase-delta. In a prospective group of uniformly handled, formalin-fixed malignant lymphomas we previously demonstrated 19A2 to be a reliable marker of proliferative activity similar to Ki-67 in frozen tissue. The present study examines the applicability of this technique in archival formalin-fixed material. Studies on tonsilar tissue revealed that formalin fixation beyond 30 hours adversely affected reactivity of 19A2, possibly explaining the variable results in nonuniformly fixed archival material. We found that only 27 (56%) of 48 archival cases of infiltrating ductal carcinoma showed sufficient reactivity with 19A2 to permit reliable quantification of the tumor growth fraction. Acid pretreatment with 2N HCl had no apparent effect on 19A2 reactivity. Using both antibodies on a group of 32 archival lymphomas, carcinomas, and sarcomas, significantly more biopsies stained reliably for PC10 (84%) than for 19A2 (72%; P < 0.036). Further, none of the cases that did not react with PC10 reacted with 19A2. PC10 may recognize a different epitope of PCNA/Cyclin which may be more resistant to alterations by fixation. In the 23 cases that reliably stained for both markers, largely carcinomas, there was excellent correlation between estimated growth fractions (r = 0.96). Although immunostaining provides a useful way to estimate tumor growth fractions in paraffin-embedded tissues, modifications of technique and cautious interpretation of results are advisable when using archival material.
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PMID:Estimation of tumor growth fractions in archival formalin-fixed, paraffin-embedded tissues using two anti-PCNA/Cyclin monoclonal antibodies. Factors affecting reactivity. 128 22

DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase alpha (pol-alpha), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, proliferating cell nuclear antigen, and DNA polymerase delta, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, indicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonucleotide synthesis, pol-alpha extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. T7 DNA polymerase was also shown to rapidly extend oligoribonucleotide primers in the presence of aphidicolin or antibodies against pol-alpha, conditions under which pol-alpha was markedly inhibited. These findings suggest that interactions between T antigen, pol-alpha-primase, and HSSB position the pol-alpha-primase complex on the lagging-strand template for RNA primer synthesis.
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PMID:Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation. 131 May 41

The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase alpha by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 microM) inhibitory to DNA polymerases beta and gamma, however, higher concentrations of ddTTP (200 microM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase epsilon (PCNA-independent DNA polymerase delta) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.
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PMID:Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replication in vitro. 131 27

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.
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PMID:DNA polymerase-delta from the silk glands of Bombyx mori. 132 38

We have used the antisense strategy to study the role of certain genes in cell cycle progression. In particular, we used antisense oligodeoxynucleotides to study: (1) the role of the IGF-1 receptor in the control of cell proliferation; and (2) the sequence of gene expression during the cell cycle. Our results can be summarized as follows: (1) the activation of the IGF-1 receptor by its ligand, IGF-1, is an obligatory step in the proliferation of fibroblasts and hemopoietic cells; and (2) the expression of DNA synthesis genes, such as PCNA, DNA polymerase alpha, and cdc2, is dependent on the expression of previous genes. A tentative temporal order is: c-myc > c-myb > IGF-1 receptor > DNA synthesis genes.
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PMID:Inhibition of cell cycle progression by antisense oligodeoxynucleotides. 134 Jan 57

The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y.S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta subunit in lieu of T4 gp45 in the elongation of singly primed phi X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.
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PMID:The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems. 134 4

The crystal structure of the beta subunit (processivity factor) of DNA polymerase III holoenzyme has been determined at 2.5 A resolution. A dimer of the beta subunit (M(r) = 2 x 40.6 kd, 2 x 366 amino acid residues) forms a ring-shaped structure lined by 12 alpha helices that can encircle duplex DNA. The structure is highly symmetrical, with each monomer containing three domains of identical topology. The charge distribution and orientation of the helices indicate that the molecule functions by forming a tight clamp that can slide on DNA, as shown biochemically. A potential structural relationship is suggested between the beta subunit and proliferating cell nuclear antigen (PCNA, the eukaryotic polymerase delta [and epsilon] processivity factor), and the gene 45 protein of the bacteriophage T4 DNA polymerase.
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PMID:Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. 134 52

Chronic lymphocytic leukemia (CLL) is a usually indolent disease that can assume an aggressive clinical course in some patients. To develop assays that would be predictive of how a particular patient's disease would evolve, we studied the expression of proliferating cell nuclear antigen (PCNA) by Western blotting in 40 patients with CLL. The concentration of PCNA, a cofactor for delta DNA-dependent DNA polymerase, is indicative of the proliferative state of the cell. Significantly lower PCNA levels were observed in earlier stage CLL when compared with more advanced disease. The leukemic cell proliferative rate, assessed by lymphocyte doubling time and flow cytometry, also correlated significantly with the level of PCNA expression. These results suggest that a high level of PCNA in the cells of CLL patients at presentation identifies a subgroup of patients whose CLL cells have a higher proliferative activity and who may, therefore, have a potentially shorter survival.
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PMID:Prognostic value of proliferating cell nuclear antigen expression in chronic lymphoid leukemia. 135 Feb 26

The elongation of primed DNA templates by DNA polymerase delta and DNA polymerase epsilon requires the action of two accessory proteins, proliferating cell nuclear antigen and activator 1 (A1, also called replication factor C). A1 is an enzyme that contains five different subunits (145, 40, 38, 37, and 36.5 kDa). In this paper, we describe the isolation of the gene encoding the 37-kDa subunit from HeLa cells. This gene was cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence shows a high degree of homology to the 40-kDa subunit of A1; they both contain the identical ATP-binding motif, but in contrast to the bacterial expressed 40-kDa protein, the 37-kDa expressed protein did not bind ATP. Both the 37- and 40-kDa proteins share substantial homology with the phage T4 gene 44 protein and to a lesser extent with the tau and gamma subunits of the E. coli DNA polymerase III holoenzyme. Polyclonal antibodies against the bacterially expressed 37- and 40-kDa proteins do not crossreact and are specific in their interaction. Antibodies against the 37-kDa protein maximally inhibited (by 50%) the A1-dependent synthesis of DNA by DNA polymerase delta; antibodies against the 40-kDa protein quantitatively inhibited the same reaction. When A1-dependent synthesis of DNA was partially inhibited by antibodies against the 40-kDa subunit, the addition of antibodies against the 37-kDa subunit inhibited DNA synthesis to a greater extent than the anti-37-kDa antibody alone. These results suggest that both the 37- and 40-kDa subunits of A1 are required for the biological role of A1 and that they may function differently in this process.
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PMID:Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells. 135 77

In eukaryotic cells, nucleus-cytoplasm exchanges play an important role in genomic regulation. We have analyzed the localization of four nuclear antigens in different growth conditions: two replicative proteins, DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), and two oncogenic regulatory proteins, c-Myc and c-Fos. A kinetic study of subcellular localization of these proteins has been done. In cultures in which cells were sparse, these proteins were detected in the nucleus. When proliferation was stopped by the high density of culture cells or by serum starvation, these proteins left the nucleus for the cytoplasm with different kinetics. DNA polymerase alpha is the first protein to leave the nucleus, with the PCNA protein, c-Fos, and c-Myc leaving the nucleus later. In contrast, during serum stimulation c-Fos and c-Myc relocalize into the nucleus before the replicative proteins. We also noticed that in sparse cell cultures, 10% of the cells exhibit a perinuclear staining for the DNA polymerase alpha, PCNA, and c-Myc proteins but not for c-Fos. This peculiar staining was also observed as an initial step to nuclear localization after serum stimulation and in vivo in Xenopus embryos when the G1 phase is reintroduced in the embryonic cell cycle at the mid-blastula stage. We suggest that such staining could reflect specific structures involved in the initiation of the S phase.
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PMID:Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression. 135 52


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