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  • 1
    Electronic Resource
    Electronic Resource
    A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro (1990)
    s.l. : American Chemical Society
    Biochemistry 29 (1990), S. 6362-6374 
    Details
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1021/bi00479a004
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Ara-C affects formation of cancer cell DNA synthesome replication intermediates (2000)
    Springer
    Cancer chemotherapy and pharmacology 45 (2000), S. 312-319 
    Details
    ISSN: 1432-0843
    Keywords: Key words Ara-C ; DNA synthesome ; DNA replication ; In vitro
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Purpose: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468. Methods: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed using neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases α and δ). Results: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 μM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase α enzyme while the DNA polymerase δ seems to be resistant to the inhibitory effect of that drug. Conclusions: Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase α and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002800050046
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Effects of gemcitabine and araC on in vitro DNA synthesis mediated by the human breast cell DNA synthesome (2000)
    Springer
    Cancer chemotherapy and pharmacology 45 (2000), S. 320-328 
    Details
    ISSN: 1432-0843
    Keywords: Key words AraC ; Gemcitabine ; In vitro ; DNA replication ; DNA synthesome
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Purpose: Gemcitabine (dFdC) and cytarabine (araC) are both analogs of deoxycytidine. Gemcitabine is a relatively new drug that has been shown in both clinical trials and in vitro systems to have more potent antitumor activity than araC. We have previously isolated a fully functional multiprotein DNA replication complex from human cells and termed it the DNA synthesome. Using the DNA synthesome, we have successfully examined the mechanism of action of several anticancer drugs that directly affect DNA synthesis. In this study, we compared the effects of dFdC and araC on in vitro DNA synthesis mediated by the DNA synthesome with the effects of these drugs on intact MCF7 cell DNA synthesis. Methods: We examined the effects of dFdC and araC on intact MCF7 cell DNA synthesis and clonogenicity. We also performed in vitro SV40 replication assays mediated by the MCF7 cell-derived DNA synthesome in presence of dFdCTP and araCTP. The types of daughter molecules produced in the assay were analyzed by neutral and alkaline agarose gel electrophoresis. Finally, we examined the effects of dFdCTP and araCTP on the synthesome-associated DNA polymerase α and δ activities. Results: Our results showed that dFdC was more potent than araC at inhibiting intact MCF7 cell DNA synthesis and clonogenicity. [3H]Thymidine incorporation was inhibited by 50% at a dFdC concentration of 10 μM, which was about tenfold lower than the concentration of araC required to inhibit intact cell DNA synthesis by the same amount. As examined by clonogenicity assay, dFdC was also significantly more cytotoxic than araC after a 24-h incubation. In vitro SV40 replication assays using the DNA synthesome derived from MCF7 cells demonstrated that the formation of full-length DNA along with replication intermediates were inhibited by dFdCTP in a concentration-dependent manner. Full-length DNA was produced in the in vitro DNA replication assay even when the dFdCTP was incubated in the assay at concentrations of up to 1 mM. We observed that in the presence of 10 μM dCTP, 3 μM dFdCTP and 60 μM araCTP were required to inhibit in vitro SV40 DNA synthesis by 50%. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there was no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome-associated DNA polymerases α and δ. It was found that the drug concentrations required to inhibit 50% of the synthesome-associated DNA polymerase δ activity were much higher than those required to inhibit 50% of DNA polymerase α activity for both dFdCTP and araCTP. Conclusion: Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase α activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002800050047
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    Paper (German National Licenses)
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  • 4
    Electronic Resource
    Electronic Resource
    Further characterization of the human cell multiprotein DNA replication complex (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 91-107 
    Details
    ISSN: 0730-2312
    Keywords: mammalian cell ; DNA replication ; multiprotein complex ; simian virus 40 ; HeLa cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase α, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374.]In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase δ, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells. © 1995 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590111
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  • 5
    Electronic Resource
    Electronic Resource
    DNA replication machinery of the mammalian cell (1998)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 72 (1998), S. 18-29 
    Details
    ISSN: 0730-2312
    Keywords: mammalian DNA replication fork ; DNA synthesome ; PCNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The process of DNA replication in mammalian cells is highly complex and has several unique features that distinguish it from simpler prokaryotic systems. The study of mammalian DNA replication lagged behind that of prokaryotes for many years. This was because of the lack of a reliable and efficient mammalian cell-based in vitro DNA replication system. In 1984, the first mammalian-based DNA replication system that initiated DNA synthesis successfully in vitro was developed. The employment of the mammalian in vitro DNA replication system has led to the identification of several DNA replication proteins. This article describes the current knowledge regarding the proteins mediating mammalian DNA replication, as well as how they are proposed to function during DNA synthesis. There is also a discussion of the role the mammalian cell nuclear architecture plays in DNA replication. The evidence for the existence of an organized DNA replication machine in mammalian cells is also presented. J. Cell. Biochem. Suppls. 30/31:18-29, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 259-267 
    Details
    ISSN: 0730-2312
    Keywords: native immunoblotting ; SV40 ; DNA replication ; in vitro ; multiprotein complexes ; in situ denaturation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Increasing evidence has supported the concept that many of the enzymes and factors involved in the replication of mammalian DNA function together as a multiprotein complex. We have previously reported on the partial purification of a multiprotein form of DNA polymerase from human HeLa cells shown to be fully competent to support origin-specific large T-antigen-dependent simian virus 40 (SV40) DNA replication in vitro. In an attempt to more definitively identify the complex or complexes responsible for DNA replication in vitro, partially purified human HeLa cell protein preparations competent to replicate DNA in vitro were subjected to native polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The Native Western blots were probed with a panel of antibodies directed against proteins believed to be required for DNA replication in vitro. Apparent complexes of 620 kDa and 500 kDa were identified by monoclonal antibodies directed against DNA polymerase α and DNA polymerase δ, respectively.To detect epitopes possibly unexposed within the native multiprotein complexes, blots were also analyzed following denaturation in situ following treatment with detergent and reducing agent. The epitope or access to the epitope recognized by the monoclonal antibody against DNA polymerase α was destroyed by exposure of the blots to denaturing conditions. In contrast, an epitope present on a very large complex of approximately 1000 kDa was recognized by a monoclonal antibody against proliferating cell nuclear antigen only following treatment of the native immunoblots with denaturing agents. Identification of these complexes will allow their further purification, characterization, and elucidation of their role in the replication of DNA. © 1996 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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