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)

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.
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PMID:Mammalian DNA nucleotide excision repair reconstituted with purified protein components. 769 16

In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment. Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA). To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions. Purified DNA polymerase delta or epsilon, with DNA ligase, was then used to form the repair patch. DNA polymerase delta could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles. The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products. DNA polymerase epsilon, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles. Addition of replication protein A changed the situation dramatically, and synthesis by polymerase epsilon became dependent on both PCNA and replication factor C. A combination of DNA polymerase epsilon, PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches.
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PMID:Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA. 771 Oct 23

The macrolide rapamycin arrests T lymphocytes stimulated by interleukin-2 (IL-2) at G1/S. We have recently found that IL-2 induced an increase in the binding of discrete transcription factors of the ATF/cAMP-responsive element binding factor (CREB) family at G1/S, and that this effect was inhibited by rapamycin (Feuerstein, N., Huang, D., Hinrichs, S. H., Orten, D. J., Aiyar, N., and Prystowsky, M. B. (1995) J. Immunol. 154, 68-79). We now show, by using high resolution two-dimensional gel electrophoresis, that rapamycin inhibited selectively the synthesis of three discrete IL-2-induced soluble proteins (35 kDa/pI approximately 5, 68 kDa/pI approximately 4, 110 kDa/pI approximately 4.3). Analysis of nuclear proteins demonstrated that rapamycin selectively blocked the expression of proliferating cell nuclear antigen (PCNA), an obligate cofactor of DNA polymerase-delta, an important component for DNA replication. Rapamycin inhibited the IL-2-induced PCNA mRNA, and the murine PCNA promoter activity in IL-2-stimulated cells. Inducible CRE-binding proteins were shown previously to be required for PCNA promoter activity in IL-2-stimulated T lymphocytes. Using DNA binding gel mobility shift assay we demonstrated that rapamycin potently inhibited the binding of CREB/ATF transcription factors to CRE elements in the murine proximal PCNA promoter. These results suggest that PCNA is a preferred target in a rapamycin-sensitive transduction pathway, and that the mechanism by which rampamycin inhibits PCNA gene expression may involve the inhibition of the interaction of CREB/ATF transcription factors with CRE elements in the proximal PCNA promoter.
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PMID:Rapamycin selectively blocks interleukin-2-induced proliferating cell nuclear antigen gene expression in T lymphocyte. Evidence for inhibition of CREB/ATF binding activities. 772 72

We have examined the capacity of calf thymus DNA polymerases alpha, beta, delta, and epsilon to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases alpha, delta, and epsilon was blocked at the base preceding the lesion. Addition of proliferating cell nuclear antigen to DNA polymerase delta and replication protein A to DNA polymerase alpha did not restore their capacity to elongate past the adduct. On the other hand, DNA polymerase beta efficiently bypassed the cisplatin adduct. Furthermore, we observed that DNA polymerase beta was the only polymerase capable of primer extension of a 3'-OH located opposite the base preceding the lesion. Likewise, DNA polymerase beta was able to elongate the arrested replication products of the other three DNA polymerases, thus showing its capacity to successfully compete with polymerases alpha, delta, and epsilon in the stalled replication complex. Our data suggest (i) a possible mechanism enabling DNA polymerase beta to bypass a d(GpG)-cisplatin adduct in vitro and (ii) a role for this enzyme in processing DNA damage in vivo.
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PMID:DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene. 777 11

Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha 180-kDa catalytic subunit gene contain a common 8 base pair (bp) promoter element, 5'-TATCGATA (DRE, Drosophila DNA replication-related element). We have generated various base substitutions and internal deletions in and around DRE (nucleotide positions -93 to -100 with respect to the transcription initiation site) of the PCNA gene in vitro and subsequently examined their effects on the binding to DREF (DRE-binding factor) and PCNA gene promote activity in cultured Drosophila Kc cells as well as in living flies. Gel mobility shift assays using nuclear extracts of Kc cells with and without competitor DNA fragments carrying the mutations indicated that the 10-bp sequence from positions -91 to -100 is essential for complex formation with DREF. Transient expression assays of chloramphenicol acetyl-transferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying the mutations revealed that the 8-bp sequence from -93 to -100 is essential for activation of the promoter in Kc cells. Examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying the mutations, introduced into flies by germ-line transformation, revealed that the 8-bp sequence is also important for DRE function during development. However, we obtained two exceptional mutations in the 8-bp sequence that did not or only marginally affected the PCNA gene promoter activity in transgenic flies. Both of these mutations effectively reduced the promoter activity in CAT transient expression assay in Kc cells and the binding to DREF in vitro. Therefore, the 8-bp sequence requirement for DRE function appears to be less stringent in living flies than in the cultured cell or in vitro cases.
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PMID:A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNa replication-related genes. 779 83

The influence of poly (ADP-ribose) polymerase (PARP) and poly ADP-ribosylation on DNA synthesis supported by human replicative DNA polymerase (DNA pol) alpha, delta, and epsilon has been examined using the replication system containing poly(dA)4500-oligo(dT)12-18 as the template primer. PARP alone inhibited the pol activities in a dose-dependent manner even in the presence of the accessory factors for DNA pol delta, proliferating cell nuclear antigen (PCNA) and activator 1 (Al; RF-C). Both DNA pol alpha and epsilon activities were decreased approximately 10-fold under the poly ADP-ribosylating condition. In contrast, DNA synthesis by DNA pol delta holoenzyme was not affected by poly ADP-ribosylation like prokaryotic DNA pol's. The analysis of poly(dT) formed by DNA pol alpha and epsilon indicated that poly ADP-ribosylation mainly reduced the frequency of replication. These observations suggest a possibility that PARP acts as a negative regulator for the initiation of DNA replication upon cellular DNA damage.
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PMID:Poly (ADP-ribose) polymerase inhibits DNA replication by human replicative DNA polymerase alpha, delta and epsilon in vitro. 780 50

Kinetic analysis of DNA polymerase epsilon in its interaction with the homopolymeric template-primer poly(dA)/oligo(dT) and a singly-primed synthetic oligonucleotide of defined sequence indicated that primer utilization is inhibited by single-stranded DNA. Long single-stranded DNA regions appear to sequester DNA polymerase epsilon via nonproductive binding, thus reducing its catalytic efficiency. Proliferating cell nuclear antigen can reduce this nonproductive effect by increasing the rate of primer binding by DNA polymerase epsilon. Once the complex between DNA polymerase epsilon and the primer is formed, proliferating cell nuclear antigen can increase the rate of nucleotide incorporation. The results suggested a dual role of proliferating cell nuclear antigen in stimulating the activity of DNA polymerase epsilon, namely, first to facilitate primer binding and second to stimulate the synthetic activity itself. A model for the interaction between these two proteins in DNA synthesis is discussed.
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PMID:DNA polymerase epsilon interacts with proliferating cell nuclear antigen in primer recognition and elongation. 782 47

Proliferating cell nuclear antigen, an auxiliary protein of deoxyribonucleic acid polymerase-delta, has been shown to be a reliable marker of nuclear deoxyribonucleic acid synthetic activity. We applied a monoclonal antibody to proliferating cell nuclear antigen to a series of serial stereotactic biopsies from patients with glioblastoma multiforme and found the proliferative activity to vary relative to biopsy location within or surrounding the solid tissue component of the tumor. Twenty-seven trajectories in 26 patients were analyzed, each consisting of two to five sequential 10 x 1.5 mm core biopsies (mean = 3). The proliferative index (PI) was greatest in those cells located at the solid tumor-infiltrated parenchyma interface. PI values were significantly lower in those biopsy cores located proximal (within infiltrated parenchyma) and distal (within solid tumor tissue) to the solid tumor-infiltrated parenchyma interface (median PI values, proximal to distal: 0.38, 0.66, 5.45 solid tumor-infiltrated parenchyma interface], 0.39, 0.09%). The mean PI values were significantly lower in neoplastic cells samples from regions of peripheral hypodensity on computed tomographic scans compared with those sampled from contrast-enhancing regions (0.9 and 3.91%, respectively). There was no significant difference in the mean PI values of neoplastic cells sampled from regions of contrast enhancement or central hypodensity (3.91 and 4.31%, respectively).
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PMID:Changes in proliferating cell nuclear antigen expression in glioblastoma multiforme cells along a stereotactic biopsy trajectory. 788 47

The determination of the structure of the processivity factor (beta subunit) of Escherichia coli DNA polymerase III holoenzyme showed that this protein acts to clamp the polymerase onto DNA by forming a closed circular structure that can encircle duplex DNA (X.-P. Kong, R. Onrust, M. O'Donnell & J. Kuriyan. (1992). Cell, 69, 425-437). In this review we describe the features of the beta subunit that allow it to be linked tightly but non-specifically to DNA, and discuss the surprisingly symmetrical architecture of the molecule. The simple repeating pattern of the chain fold allows a connection to be made to the as yet unknown structures of eukaryotic proliferating cell nuclear antigen and the gene 45 protein of bacteriophage T4, which are the processivity factors of the corresponding DNA polymerases.
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PMID:Sliding clamps of DNA polymerases. 790 1

To identify the preneoplastic lesions of hepatocellular carcinoma and the fine structure of preneoplastic hepatocytes, we studied proliferative conditions in cirrhosis of the liver. In all, 46 foci of cellular alteration (FCA), three regions of adenomatous hyperplasia (ADH), and 21 small hepatocellular carcinomas (sHCC) were studied by published criteria for sHCC and by the proliferative activity of the lesions as examined with monoclonal antibodies against DNA polymerase alpha and proliferating cell nuclear antigen. The four patients with FCA composed of basophilic hepatocytes were classified by the criteria as having sHCC; cells had features similar to those of sHCC. Two of these four patients with FCA were found to have HCC several years later. The number of hepatocytes stained for proliferating cell nuclear antigen was 72 and 81 per 1000 hepatocyte nuclei in the two patients who developed HCC. In one of the three patients with ADH, a sHCC was found 1 year later, and dysplastic hepatocytes from the region of ADH in this patient had features similar to those of HCC cells by light and electron microscopy. In this patient, the number of hepatocytes stained for DNA polymerase alpha was 452 per 1000 nuclei. Therefore, FCA and ADH might be preneoplastic lesions of sHCC in cirrhosis of the liver. Preneoplastic hepatocytes seem to be small cells with basophilic cytoplasm, with a large nucleus to cytoplasm ratio, finely indented nuclei with a smaller amount of condensed chromatin than normal, and poorly to moderately developed organelles.
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PMID:Detection of the preneoplastic lesions of small hepatocellular carcinoma in cirrhotic livers. 790 90


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