Analysis of Covalent Complexes Formed between Calf Thymus DNA Topisomerase and Single-Stranded DNA

Prell, Brigitte; Vosberg, Hans−Peter

doi:10.1111/j.1432-1033.1980.tb04734.x

Cited by 64 publications

(34 citation statements)

References 38 publications

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“…Holden and R. Low, manuscript submitted). This difference in template preference may account for the finding that SS DNA is a more potent inhibitor of the bacterial relaxation activity than that of the eucaryotic enzyme (11). Studies of the cleavage of SS DNA using SS and gapped templates also indicate that the procaryotic and eucaryotic enzymes prefer quite different cleavage sites in terms of sequence and potential secondary structure (6,(13)(14)(15)(16)(17).…”

Section: Introductionmentioning

confidence: 94%

“…The eucaryotic enzymes relax both negatively-and positively-supercoiled DNAs to completion (7) while the bacterial enzymes fail to relax positively-twisted DNAs and only partially relax negatively-supercoiled templates (8). During catalysis, the bacterial enzymes form a covalent intermediate with the 5'-phosphoryl at the site of strand cleavage (9) while the eucaryotic enzymes attach via the 3'-phosphoryl (10,11). The bacterial enzymes show a strong preference for catalysis in regions of single-stranded (SS) DNA.…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate

Low

Holden

1985

Nucl Acids Res

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The relaxation activity of DNA topoisomerase I from HeLa cell nuclei is strongly inhibited by a variety of purine nucleotides in the presence but not absence of 1 mM potassium phosphate. For ATP, 3-4 nM causes nearly complete inhibition. The 2'-and 3'-AMP isomers are active as well in the presence of 1 mM phosphate, but the 5'-AMP isomer and adenosine are inert. At 3 nM ATP, the titration curve for phosphate is sigmoidal with inhibition beginning abruptly at about 0.5 nM. The negatively-supercoiled DNA isolated from an "inhibited" reaction is relaxed as well as the standard DNA template in the absence of ATP and phosphate suggesting that inhibition does not result from an alteration of the template which protects against its relaxation. Relaxation of positively-supercoiled DNA is also inhibited.Catalysis by E. coli DNA topoisomerase I and HeLa DNA topoisomerase II is not inhibited at concentrations of ATP and phosphate sufficient to cause 80-90% inhibition of HeLa type 1 enzyme.

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Section: Introductionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate

Low

Holden

1985

Nucl Acids Res

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“…It has been proposed that the resealing step cannot efficiently occur with denatured D N A since nicking is followed by free diffusion of contiguous D N A ends (Liu, 1983). Moreover, the eukaryotic enzyme is bound to the 3' phosphoryl end at the site of the nick in single-stranded D N A (Prell & Vosberg, 1980;Halligan et al, 1982;Trask & Muller, 1983). The virion topoisomerase also displays this characteristic.…”

Section: The Virion-associated Topoisomerase I Stability Binds To 3' mentioning

confidence: 99%

“…In addition, type II topoisomerases will decatenate interlocking DNA rings (Marini et al, 1980). Topoisomerases are characterized by their ability to form stable (probably covalent) bonds with DNA during the reaction sequence (Prell & Vosberg, 1980;Sander & Hsieh, 1983;Trask et al, 1984). Indeed, this feature led to the development of an assay which detects the covalent DNA/topoisomerase intermediate (Trask et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

Association of Type I DNA Topoisomerase with Herpes Simplex Virus

Muller¹,

Bolles²,

Parris

1985

Journal of General Virology

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SUMMARYA topoisomerase activity is associated with herpes simplex virus type 1. The enzyme was recovered from purified virions which were disrupted with 6 M-guanidine HC1 followed by renaturation of extracted proteins. Based upon the following observations, the virion activity is classified as a type I topoisomerase: (i) the linking number of a unique DNA topoisomer is altered in steps of one; (ii) ATP and MgC12 are not required for activity; (iii) the enzyme can be trapped in a covalent complex with DNA; (iv) the covalent linkage to DNA is through a 3' phosphoryl bond. A number of lines of evidence strongly indicate that the topoisomerase is external to the nucleocapsid. For example, the activity was released by treatment of intact virions with NP40, and subsequent washing steps extracted most residual activity. When guanidine extracts were prepared from nucleocapsids, topoisomerase activity was not detectable. Finally, DNA within the virion did not appear to contain covalently attached proteins with properties similar to topoisomerases. Thus, the enzyme appears to be a component of the envelope or tegument structure of the virion.

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“…Topo I activity was measured by the relaxation ofsuperwiled pIas_ mid DNA (pUCl9) [5]. One unit was defined as the amount of cnzymc that relaxes half of the added plasmid DNA (1 pg) in IO min at 25%.…”

Section: Other Proceduresmentioning

confidence: 99%

Two subforms of eukaryotic topoisomerase I Purification and structure‐function relationships

et al. 1992

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A new method for isolation ofeukaryotic topoisomcrase 1 from calf thymusand from Jurkat-l cells using HPLC has been developed. The method allows quantitative purificntion of high molLvular weight topo I and of two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions bc characterized as two subforms of the enzyme possessing structural and functional diffcrcnccs. The differences in their specific activities, sensitivity to camptothccin and in their protcolytic digestion maps have been demonstrated for the two cnzymcs.Topoisomerasc I; Subform; HPLC; Hydrophobic interaction chromatography; Camptotbecin rcsistancc lNTRODUCTlONThe type I topoisomerases (topo I) are key er~qmcs in the control of DNA topological state changes neccssary for many genetic processes [l].The procedure for isolation of topo I from calf thymus [2] and some other tissues [3] yields several polypeptides of different molecular weight. The same phenomenon was found in prokaryotic cells [4]. This might be due to partial proteolysis of the native topo I of high molecular weight. Here we suggest that this phenomenon can be caused also by another reason. EXPERIMENTAL PtwiJiccttion of type I ropoisottwoscTopo 1 was initially purified from calf thymus and Jurkat-I cells as described [3]. Then we developed our original HPLC protocol. The Altex (Beckman, Model 332) HPLC system WBS used. All steps were carried out at 4OC with buffers containing 1.4 mM 2-mercaptorthanol (M)3) and I mM PMSF; the flow rate was 0.5 ml/min.The active fraction cluting from a heparin-Scpharose column was dilutLti to a concentration of 5% glycerol. Solid ammonium sulfate (AS) was slowly added to 33% saturation, and after stirring for GO min the precipitate was removed by ccnlrifugation (I 3,000 x 6, I5 min). The supernatnnt was applied to Phenyl SPW (TSK column), cquili- bratcd with buffer A, containing 50 mM potassium phosphate buffer (KPB), pH 7.3. and 33% saturation with AS. Elution was performed with an increasing linear gradient of buffer B, containing 50 mM KPB, pH 7.2. and 30% cthylcnc glycol. After dialysis against 50 mM KPB, pH 7.2, and 20% glycerol (buffer C), specific topo I activity was found in the bound and unbound fractions. The last was loaded onto a Mono Q column, and the unbound fraction from the Mono Q coiumn was then loaded directly onto a Mono S column: at this point all the activity was bound to the column. Both columns were ctquilibratcd with buffer C and in both cases the elution was performed with aa increasing linear gradient of 50 mM KPB, pH 7.2, 20% glycerol and 1 M N&l (buffer D). Other proceduresTopo I activity was measured by the relaxation ofsuperwiled pIas_ mid DNA (pUCl9) [5]. One unit was defined as the amount of cnzymc that relaxes half of the added plasmid DNA (1 pg) in IO min at 25%. The assay was also carried out with eamptothccin (CPT) at different concentrations. Isoelectric focusinfi with subseauent immunodctcc-

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Analysis of Covalent Complexes Formed between Calf Thymus DNA Topisomerase and Single-Stranded DNA

Cited by 64 publications

References 38 publications

Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate

Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate

Association of Type I DNA Topoisomerase with Herpes Simplex Virus

Two subforms of eukaryotic topoisomerase I Purification and structure‐function relationships

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