Replication of E. coli duplex DNA in vitro

Nüsslein, Volker; Henke, Sigrid; Johnston, Leland H.

doi:10.1007/bf00269592

Cited by 8 publications

(1 citation statement)

References 16 publications

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“…It is clear from both in vivo and in vitro studies that dnaB protein, in particular, acts at replication forks of the chromosome (16,(20)(21)(22). Its function in host replication may be similar to that in replication of 6X174 DNA in vitro.…”

Section: Ox174 Replication Intermediate Contains Dnab Proteinmentioning

confidence: 99%

Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication

McMacken

Ueda

Kornberg

1977

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

137

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The first step in conversion of 4X174 singlestranded DNA to the duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, L. Rowen and A. Kornberg, unpublished data).In the present work, dnaB protein has been shown to be an -active component of the 4X174 replication intermediate; it sustains synthesis by primase of a ribonucleotide primer fragment when coupled to replication, and multiple primers on the OX174 circle when uncoupled from it. Our findings suggest a mechanism whereby dnaB protein utilizes its ATPase activity (11) to propel itself along the DNA strand, thus serving as a mobile promoter or recognition signal for the action of primase. MATERIALS AND METHODSMaterials. Buffer A contained 10 mM imidazole-HCI (pH 7.0), 5% (wt/vol) sucrose, 20 mM KCl, 10 mM dithiothreitol, 0.1 mM ATP, 10 mM MgCl2, and 70 Mg of bovine serum albumin per ml. Buffer B contained 120 mM MgCl2, 20mM ATP, 40 mM spermidine-HCI, and 40 mM Tris (pH 7.5). Buffer C contained 50mM Tris-HCl (pH 7.5), 10% (wt/vol) sucrose, 20 mM dithiothreitol, and 200 ,ug of bovine serum albumin per ml. Buffer D contained 10 mM Tris-HCI (pH 8.0), 0.1 M NaCl, and 1 mM EDTA.[y-32P]ATP was synthesized by the procedure of Maxam and Gilbert (12). The extensively purified Escherichia coli replication proteins used in this work will be described elsewhere. Antibodies against dnaB protein and protein n, and other materials and reagents, were prepared as described (3, 4).Formation and Isolation of Replication Intermediate. The replication intermediate was formed and assayed essentially as described (4). It was isolated free of unassociated protein by filtering the reaction mixture through Bio-Gel A-15m agarose (equilibrated in buffer A at 250) and collecting the void (excluded) volume.Assay of RNA Primer Synthesis. A detailed report on RNA primer synthesis on OX 174 DNA will be published elsewhere.Briefly, the components used for formation of 1.1 nmol of replication intermediate (4)

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Section: Ox174 Replication Intermediate Contains Dnab Proteinmentioning

confidence: 99%

Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication

McMacken

Ueda

Kornberg

1977

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

137

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In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication

Nüsslein-Crystalla

Scheefers-Borchel

1979

Molec. Gen. Genet.

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The restriction nuclease cleavage pattern of E. coli DNA synthesized in vitro in the cellophane membrane system (Schaller et al., 1972) is similar to the one obtained after labelling E. coli in vivo. This is shown for exponentially growing cells and for cells synchronized by amino acid starvation followed by thymine starvation. In synchronized cells a piece of some 180 kilobase pairs is labelled containing oriC and neighbouring regions at 82 min on the genetic map of E. coli. A pulse label in vitro is incorporated into the same piece of DNA, but the center of this region, i.e. the EcoR1 fragment of 8.6 kbp length which contains the oriC region (Marsh and Worcel, 1977; v. Meyenburg et al., 1977; Yasuda and Hirota, 1977) is missing.

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In vitro system for the replication of the mini R1 factor Rsc11

Bezanson

Goebel

1979

Molec. Gen. Genet.

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Replication of E. coli duplex DNA in vitro

Cited by 8 publications

References 16 publications

Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication

Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication

In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication

In vitro system for the replication of the mini R1 factor Rsc11

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