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Unformatted text preview: Escherichia coli Also Lacking DNA Polymerase Activity Deoxynucleoside Triphosphates by DNA Thermosensitive Mutants of On the Process of Cellular Division in Escherichia coli, V. Incorporation of Jose Mordoh, Yukinori Hirota, and Francois Jacob doi:10.1073/pnas.67.2.773 1970;67;773-778 PNAS This information is current as of January 2007. www.pnas.org#otherarticles This article has been cited by other articles: E-mail Alerts . click here article or article - sign up in the box at the top right corner of the Receive free email alerts when new articles cite this Rights & Permissions www.pnas.org/misc/rightperm.shtml entirety, see: To reproduce this article in part (figures, tables) or in Reprints www.pnas.org/misc/reprints.shtml To order reprints, see: Notes: ProceedingsoftheNational Academy ofSciences Vol. 67, No. 2,pp. 773-778, October 1970 On theProcess ofCellular Divisionin Escherichia coli, V. Incorporation of DeoxynucleosideTriphosphates byDNA ThermosensitiveMutants ofEscherichiacoli AlsoLackingDNA PolymeraseActivity* Jose Mordoht,Yukinori Hirota, and Francois Jacob SERVICE DE. GINkTIQUE CELLULAIRE DE L'INSTITUT PASTEUR ET DU COLLIGE DE FRANCE, PARIS CommunicatedJuly28,1970 Abstract. Double mutants ofEscherichiacoliwereconstructedby combining DNA thermosensitive mutations affecting either DNA initiation (DnaA) or elongation (DnaB) withthePolAl mutation,whichabolishesDNA polymerase activity. Incorporation oflabeleddeoxynucleoside triphosphates was studied inthesemutantsusingtoluene-treatedcells. Thisincorporationwas foundtobe exactly correlatedwiththecapacityofthemutants tosynthesizeDNA invivo. The processofchromosome replicationcanbe dividedintotwosteps: initia- tionofDNA synthesisandelongationofthemolecule. Thermosensitivemutants have beenisolatedthatareunabletoperformone ortheotherprocessathigh temperature.1-4 Until recently, however, no biochemical defect, eitherinthe DNA polymerase orintheestablishment ofthe nucleotidepools,couldbecor- relatedwiththegeneticlesionofsuchmutants."2'4 Thisresult,takentogether with a varietyofargumentsindicatingthatchromosome duplicationislikelyto occur in the membrane, suggested that DNA polymerase isnot the enzyme involved in this process. This was reinforced by the isolation5 ofa mutant (W3110 P3478 Pol Al-) lackingDNA polymerase but stillableto multiply. In addition, the availability ofsuch a mutant offersa new possibilityforthe biochemical investigationofDNA thermosensitive mutants. In thispaperwe report (1)amethod fordetectingtheincorporationofdeoxynucleosidetriphos- phates (dNTPs) intoluene-treatedbacteriaand (2)thebehavioratpermissive and nonpermissive temperatures of double mutants carrying both a Pol Al mutation and a thermosensitive DNA-synthesis mutation. The method for detecting the incorporation of dNTPs is a modificationofthatdeveloped by MosesandRichardson6andSmithetal.7 Materials and Methods. Strains: The following strains were used: W3310 P3478: F- Thy- PolAl- (agiftfrom Dr.J.CairnsviaDr.F.Bonhoeffer); CRT4634:(agiftfrom Dr....
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