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

Abstract

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duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, L. Rowen and A. Kornberg, unpublished data).In the present work, dnaB protein has been shown to be an -active component of the 4X174 replication intermediate; it sustains synthesis by primase of a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)

A DNA primase specified by I-like plasmids.

“…In this respect the enzyme resembles the phage T7 gene-4 primase, which shows a similar lack of template specificity (30). In addition, whereas the dnaG primase action on 4X174 DNA is strictly dependent on accessory replication proteins such as dnaB and dnaC (22,23,31), plasmid primase has no such dependency ( Table 3). The oligonucleotide-synthesizing activity of the two priming enzymes are also different; the plasmid primase, unlike the dnaG primase, does not incorporate deoxyribonucleotides into mixed ribo-deoxyribopolymers (26,27).…”

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

“…Reconstitution of DNA replication with purified proteins has yielded great insight into the mechanism of DNA replication as well as other aspects of DNA metabolism, such as DNA repair and recombination in prokaryotic and eukaryotic cells (5)(6)(7)(8)(9)(10). The replication of the E. coli chromosome requires a large number of proteins that have to work in concert in order to successfully accomplish initiation, elongation, and termination of DNA replication (2,(11)(12)(13)(14). Thus, a careful analysis of the interactions between replication factors is of critical importance for gaining further insights into the mechanism and control of the major steps of DNA replication.…”

Movement and site selection for priming by the primosome in phage phi X174 DNA replication.

“…t An interesting comparison exists with phage T7 and phage T4 replication. The T7-encoded gene 4 protein is a helicase (33) as well as a primase (34). The T4-encoded gene 41 and 61 proteins appear to be analogous to dnaB protein and primase in facilitating the propulsion ofthe replication fork (35).…”

“…teins, SSB-coated OX DNA is activated to form a prepriming replication intermediate (3) that promotes synthesis of multiple primers by primase (4,5). Protein n' (6) and dnaB protein (7,8) are DNA-dependent ATPases and dnaC protein interacts with dnaB protein in the presence ofATP (9).…”