Solubilization and Partial Characterization of a Multienzyme Complex of DNA Synthesis from Human Lymphoblastoid Cells

Wickremasinghe, RG; Yaxley, John C.; Hoffbrand, A. V.

doi:10.1111/j.1432-1033.1982.tb06821.x

Cited by 40 publications

(20 citation statements)

References 23 publications

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“…Thymidine kinase has been detected in a nuclear, multienzyme complex involved in DNA synthesis in some mammalian cells (Reddy & Pardee, 1982;Wickremasinghe et al, 1982Wickremasinghe et al, , 1983. Among the DNA-binding proteins induced by HSV-1 is one of the same Mr (43000) as the largest TK polypeptide (Bayliss et al, 1975;Powell & Purifoy, 1976).…”

Section: Introductionmentioning

confidence: 99%

Evidence that Deletion of Coding Sequences in the 5' End of the Thymidine Kinase Gene of Herpes Simplex Virus Type 1 Affects the Stability of the Gene Products

Haarr

Flatmark²

1987

Journal of General Virology

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SUMMARYThe experiments described in the present work were designed to study the function of the N-terminal end of thymidine kinase (TK) encoded by herpes simplex virus type 1. Specifically we were interested to know whether this end was involved in binding of the enzyme to other molecules, had any influence on its subcellular localization or affected one or more of the activities associated with the enzyme. A parental enzyme and a deletion mutant, lacking the 45 N-terminal amino acids, derived from this strain, were used. Thymidine kinase from the parental virus bound to DNA-Sepharose, but the truncated enzyme did not. This was apparently not due to a specific ability to bind to DNA, since immunofluorescence studies indicated that both the normal and the deleted TK were mainly located in the cytoplasm, preferentially in the perinuclear region. Phosphorylation of thymidine as well as the amounts of TK potypeptides were markedly reduced at late times after infection with the mutant, but not to the same extent after infection with the wild-type. The deleted TK gene was efficiently transcribed as shown by hybridization of RNA to a probe specific for the gene, and this RNA directed the synthesis in vitro of TK polypeptides. Deletion of the 5' end of the gene seems to affect the stability of either the enzyme or TK-specific mRNA, or both. The TMP phosphorylating activity seems to be particularly destabilized relative to the thymidine phosphorylating activity.

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Section: Introductionmentioning

confidence: 99%

Evidence that Deletion of Coding Sequences in the 5' End of the Thymidine Kinase Gene of Herpes Simplex Virus Type 1 Affects the Stability of the Gene Products

Haarr

Flatmark²

1987

Journal of General Virology

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“…Another replication factor might be a better candidate for the target of HU. It is worth noting that recent evidence indicates that replication takes place in a multienzyme complex which appears to be extremely fragile and cannot be functionally reassembled in vitro (40). HU might interfere directly or indirectly with the formation or the stability of this complex.…”

Section: Resultsmentioning

confidence: 99%

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

Wawra

Wintersberger

1983

Mol. Cell. Biol.

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Cell-free DNA synthesis was performed in a lysed cell system from mouse cell cultures. The in vitro reaction was totally inhibited by N-ethylmaleimide but unaffected by hydroxyurea or fluorodeoxyuridine when these compounds were added to the incubation mixture. However, in a preparation obtained from cells which had been blocked by hydroxyurea before lysis, the rate of DNA synthesis was markedly reduced. This effect could not have been caused by the depletion of the precursor pools as all necessary triphosphates were added to the in vitro incubation mixture. Analysis by alkaline density gradients showed that the ligation of primary synthesis products is retarded in hydroxyurea-pretreated lysed cells and that small fragments accumulate. These results suggest that hydroxyurea interferes with the processing of early replication products, preventing the formation of longer intermediates. Its mechanism is either independent from the well-known inhibition of ribonucleoside diphosphate reductase or it may be the result of an as-yet-unknown function of this enzyme in a later step of replication. This observation could help to explain why cells appear to be blocked by hydroxyurea in the early part of the S phase (rather than at the Gl/S border proper) and also why DNA repair synthesis is relatively insensitive to the drug.Hydroxyurea (HU) is a specific inhibitor of DNA replication (34). It suppresses the polymerization of new strands almost completely and causes the accumulation of short DNA fragments that cannot be further elongated (16,21,24,25,29). Repair synthesis is much less affected by the drug (34). It has generally been assumed that the block of the conversion of ribonucleotides to deoxyribonucleotides via the inhibition of the enzyme ribonucleoside diphosphate reductase is responsible for these effects of HU (17,18,20). On the other hand, the difference in the response of replicative versus repair synthesis to HU is not obvious; the substrate affinities of the known DNA polymerases do not vary enough to provide a satisfactory rationale (4, 6). Moreover, the proposed mechanism cannot explain why HU-blocked cells do not stop at the Gl/S boundary but start replication and accumulate in the early S phase (10, 36).The observation that, in a cell-free DNAsynthesizing system obtained from HU-treated cells, the inhibition was still partly effective although all triphosphates were present in theincubation mixture led us to reconsider the effect of HU. This phenomenon was specific for HU and could not be observed with fluorodeoxyuridine (FUdR), another substance which interferes with the precursor pool. Hence, it is unlikely that the observed lasting inhibition is due to the interference of HU with the biosynthesis of precursors. A more detailed investigation of this effect of HU is the subject of this report. (41) and grown in plastic petri dishes at 37°C in a water-saturated atmosphere with 7.5% C02, using DME medium containing 10% calf serum. After reaching confluence, cells stopped division, and they were usually us...

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“…The subunit behavior of ribonucleotide reductase is now quite involved and a putative "replitase" complex has been described. Comprising DNA polymerase, topoisomerase, thymidylate synthetase, ribonucleotide reductase, and other enzymes concerned with DNA replication, it is proposed to form from the individual enzyme components at the end of GI and to serve to appropriately channel metabolites for DNA replication (47,48). Ribonucleotide reductase allosteric (M,) and catalytic (M2) subunits also appear to be assembled together at the G, /S interface.…”

Section: Discussionmentioning

confidence: 99%

Deoxyadenosine triphosphate as a mediator of deoxyguanosine toxicity in cultured T lymphoblasts.

Mann¹,

Fox²

1986

J. Clin. Invest.

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The mechanism by which 2'-deoxyguanosine is toxic for lymphoid cells is relevant both to the severe cellular immune defect of inherited purine nucleoside phosphorylase (PNP) deficiency and to attempts to exploit PNP inhibitors therapeutically. We have studied the cell cycle and biochemical effects of 2'-deoxyguanosine in human lymphoblasts using the PNP inhibitor 8-aminoguanosine. We show that cytostatic 2'-deoxyguanosine concentrations cause Gj-phase arrest in PNP-inhibited T lymphoblasts, regardless of their hypoxanthine guanine phosphoribosyltransferase status. This effect is identical to that produced by 2'-deoxyadenosine in adenosine deaminase-inhibited T cells. 2'-Deoxyguanosine elevates both the 2'-deoxyguanosine-5'-triphosphate (dGTP) and 2'-deoxyadenosine-5'-triphosphate (dATP) pools; subsequently pyrimidine deoxyribonucleotide pools are depleted. The time course of these biochemical changes indicates that the onset of GI-phase arrest is related to increase of the dATP rather than the dGTP pool. When dGTP elevation is dissociated from dATP elevation by coincubation with 2'-deoxycytidine, dGTP does not by itself interrupt transit from the GI to the S phase.

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Solubilization and Partial Characterization of a Multienzyme Complex of DNA Synthesis from Human Lymphoblastoid Cells

Cited by 40 publications

References 23 publications

Evidence that Deletion of Coding Sequences in the 5' End of the Thymidine Kinase Gene of Herpes Simplex Virus Type 1 Affects the Stability of the Gene Products

Evidence that Deletion of Coding Sequences in the 5' End of the Thymidine Kinase Gene of Herpes Simplex Virus Type 1 Affects the Stability of the Gene Products

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

Deoxyadenosine triphosphate as a mediator of deoxyguanosine toxicity in cultured T lymphoblasts.

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