Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate

Low, Robert L.; Holden, Joseph A.

doi:10.1093/nar/13.19.6999

Cited by 21 publications

(16 citation statements)

References 35 publications

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“…HeLa topo I and II were purified according to published procedures (13,14) except all columns were eluted with linear salt gradients. Topo I was inhibited by a factor of 50 under DNA unwinding assay conditions to be described, probably due to the inhibitory effects of ATP and creatine phosphate (15 …”

Section: Methodsmentioning

confidence: 87%

Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.

Dean¹,

Bullock²,

Murakami³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

345

226

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The simian virus 40 (SV40) large T antigen (large tumor antigen), in conjunction with a topoisomerase, a DNA binding protein, and ATP, catalyzed the conversion of a circular duplex DNA molecule containing the SV40 origin of replication to a form with unusual electrophoretic mobility that we have named form U. Analysis of this molecule revealed it-to be a highly underwound covalently closed circle. DNA unwinding was not detected with DNA containing a SV40 T-antigen binding site II mutation that renders the DNA inactive in replication. The unwinding reaction requires the action of a helicase, and SV40 T-antigen preparations contain such an activity. The T-antigen-associated ability to unwind DNA copurifiled with other activities intrinsic to T antigen [ability to support replication of SV40 DNA containing the SV40 origin, poly(dT)-stimulated ATPase activity, and DNA helicase]. However, in contrast to the unwinding activity, the SV40 T-antigen- This sequence of events is analogous to the steps proposed for the initiation of replication in prokaryotic systems. For instance, initiation of Xdv (8) and Escherichia coli origin (oriC) replication (9) is believed to depend upon precise protein interactions with the replication origin. These interactions enable the DnaB protein, a DNA helicase (10), to unwind the duplex. Furthermore, when a plasmid containing the E. coli oriC sequence was incubated with proteinsDnaA, DnaB, and DnaC, protein HU, single-strand DNA binding protein (SSB), and DNA gyrase-a DNA form of unusual electrophoretic mobility was produced (11). This form consisted of highly underwound covalently closed circles, and it was suggested that the helicase and DNA gyrase drive the unwinding.We have detected a similar origin-specific unwinding of DNA in a eukaryotic system, using SV40 origin-containing DNA. The protein requirements for the assay are T antigen, SSB, and a topoisomerase (topo) to relax the resultant supercoils. The reaction is also dependent on ATP, an ATP regenerating system, and Mg2". These observations suggested that T antigen is associated with a DNA helicase activity and that T antigen is responsible for the duplex DNA unwinding that is likely to initiate replication. While this work was in progress Stahl et al. (12) reported that SV40 T antigen contains a DNA helicase activity. MATERIALS AND METHODSAll reagents, enzyme preparations, and assay procedures used were as previously described (3) 16The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Methodsmentioning

confidence: 87%

Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.

Dean¹,

Bullock²,

Murakami³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

345

226

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“…These conditions, however, can both inhibit DNA topoisomerase I and increase DNA topoisomerase II activity. Thus, ATP and phosphate inhibit topoisomerase I (Low and Holden, 1985;Castora and Kelly, 1986) whereas phosphorylation activates topoisomerase H (Sahyoun et al, 1986). Moreover, glycerophosphate is a phosphatase inhibitor (Foulkes and Rich Rosner, 1985).…”

Section: Resultsmentioning

confidence: 99%

Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

1988

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Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes. Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+. However these factors are essential for the stability and the physiological spacing of the assembled chromatin. gamma‐S‐ATP can substitute for ATP in this process. With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process. Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II. Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA. From these results we propose that mature chromatin is formed in a two‐step reaction. In the first step, nucleosome deposition occurs independently of ATP and Mg2+. Thus, nucleosome formation can be uncoupled from their spacing. In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself. The second step, requiring ATP and Mg2+, generates properly spaced chromatin.

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“…However, it has been reported that physiological concentrations of ATP (ϳ2 mM) can inhibit human Topo I (36). In another report, ATP was shown to inhibit human Topo I only in the presence of 1 mM KPO 4 (37). These conflicting reports prompted us to examine the effect of ATP and KPO 4 on the activity of HeLa Topo I in the presence or absence of 10 mM Mg 2ϩ (Table I).…”

Section: Expression and Purification Of Recombinant Forms Of Hu-mentioning

confidence: 99%

Biochemical and Biophysical Analyses of Recombinant Forms of Human Topoisomerase I

Stewart¹,

Ireton

Parker³

et al. 1996

Journal of Biological Chemistry

136

160

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Amino acid sequence comparisons of human topoisomerase I (Topo I) with seven other cellular Topo I enzymes reveal that the enzyme can be divided into four major domains: the unconserved NH 2 -terminal domain (24 kDa), the conserved core domain (54 kDa), a poorly conserved linker region (5 kDa), and the highly conserved COOH-terminal domain (8 kDa), which contains the active site tyrosine. To investigate this predicted domain organization, recombinant baculoviruses were engineered to express the 91-kDa full-length enzyme, a 70-kDa NH 2 -terminally truncated enzyme that is missing the first 174 residues, and a 58-kDa NH 2 -and COOHterminally truncated core fragment encompassing residues 175-659. The specific activity of the full-length and , as well as the polycation spermidine, have been shown to stimulate activity (2-5). Phosphodiester bond energy is preserved during the nicking-closing cycle by the formation of a phosphotyrosine bond between the active-site tyrosine and the 3Ј-end of the broken strand (6 -8). This covalent intermediate can be trapped by denaturing the enzyme during catalysis with either SDS or alkali (9 -11). Sequence analyses of a large number of SDS-induced breakage sites indicated that the cellular Topo I enzymes will cleave at specific sequences (9, 12, 13), but there is only limited sequence similarity between such sites (9, 12-17). The SDS-induced cleavage at many breakage sites is enhanced by camptothecin, a plant alkaloid that inhibits the cellular enzymes by reversibly binding to the covalent Topo I-DNA intermediate in a manner that slows the the religation step of catalysis (17-22).The human Topo I is composed of 765 residues with a predicted molecular mass of 91 kDa. Sequence comparisons of cellular eukaryotic Topo I proteins demonstrate that the human Topo I can be divided into four domains (Fig. 1).2 Residues Met 1 -Lys 197 (24 kDa) comprise the unconserved NH 2 -terminal domain, which is highly charged (Asp ϩ Glu ϭ 27%; His ϩ Lys ϩ Arg ϭ 68%) and contains four putative nuclear localization signals (24). Residues Glu 198 -Ile 651 (54 kDa) form the conserved core domain, which is followed by a short positively charged linker domain of unconserved residues Asp 652 -Glu 696 (5 kDa). Finally, residues Gln 697 -Phe 765 (8 kDa) make up the highly conserved COOH-terminal domain, which contains the active site tyrosine at position 723 (25, 26).2 Previous reports have demonstrated that the NH 2 -terminal domain is sensitive to proteolyis (3) and that residues 1-230 can be removed with little if any consequence for Topo I activity (25, 27). In contrast, a 5-amino acid deletion from the COOH terminus abolishes activity. 3 We have used the baculovirus-infected insect cell system to overproduce full-length, as well as NH 2 -and COOH-terminal deletions of human topoisomerase I. The purified recombinant Topo I is by all biochemical criteria tested identical to the native enzyme purified from human cells. Furthermore, we find the activities of the full-length protein and an aminoterminally deleted...

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Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate

Cited by 21 publications

References 35 publications

Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.

Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.

Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

Biochemical and Biophysical Analyses of Recombinant Forms of Human Topoisomerase I

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