Escherichia coli mutants temperature-sensitive for DNA synthesis

137

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duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, L. Rowen and A. Kornberg, unpublished data).In the present work, dnaB protein has been shown to be an -active component of the 4X174 replication intermediate; it sustains synthesis by primase of a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

Section: Ox174 Replication Intermediate Contains Dnab Proteinmentioning

confidence: 99%

“…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

137

“…Many mutants impaired for either replication initiation or elongation were initially isolated based on their growth defects or an impaired ability to maintain plasmids (20)(21)(22). We reasoned that mutants impaired for the ability to complete replication might be expected to exhibit similar phenotypes and initially focused our attention on the properties of recBC and recD mutants.…”

mentioning

confidence: 99%

Completion of DNA replication in Escherichia coli

Wendel

Courcelle

2014

134

The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.replication completion | double-strand break repair | RecBCD | homologous recombination | SbcDC

“…The genes that determine these proteins have been designated dnaA (1), dnaB (2), dnaC(D) (3), dnaE (4), dnaG (3), dnafH (5), dnaH (6), dnaI (7), dnaP (8), lig (9), and polA (10). Mutants in dnaE contain thermolabile DNA polymerase III (11,12); lig mutants contain thermolabile DNA ligase (9,13,14); and polA mutants are temperature-sensitive for growth when they contain thermolabile 5' to 3' exonuclease (exonuclease VI) (15).…”

mentioning

confidence: 99%

Interaction of Escherichia coli dnaB and dnaC(D) gene products in vitro.

Wickner¹,

Hurwitz²

1975

172

(16)(17)(18)(19). Each of these dna gene products has been isolated from both wild-type and temperature-sensitive strains and the thermolability of the temperature-sensitive protein has been demonstrated in the 4X174 DNA-dependent complementation system. As yet, no enzymatic activity has been found associated with dnaG or dnaC(D) gene products. The dnaC and dnaD gene products have been shown to be identical in vitro (16) and in vivo (20); the protein will be referred to here as dnaC(D) gene product. Purified dnaB gene product contains ribonucleoside triphosphatase activity that is stimulated by single-stranded DNA. These triphosphatase activities and dnaB complementing activity copurify over the last 20-fold of a 40,000-fold purification procedure (21). The purpose of this report is to describe the physical and functional interaction of two of these proteins involved in replication, dnaB and dnaC(D) gene products.tation in the 4X174 DNA-synthesizing system. The assay conditions, definitions of units, and purification procedures were described previously for dnaB (19,21) Physical Association of dnaB and dnaC(D) Gene Products. The dnaC(D) gene product was detected (as measured by stimulation in the 4X174 DNA-dependent complementation assay) physically associated with the dnaB gene product (also measured by the OX174 DNA-dependent complementation system) when the two purified proteins were mixed and subjected to gel filtration through BioGel A-5m agarose in the presence of ATP (Fig. 1A). Incubation of dnaC(D) with dnaB gene product prior to gel filtration was not essential and did not result in an increased amount of dnaC(D) associated with dnaB. Fig. 1B Fig. lA and B were obtained when columns were developed with 50 mM Tris-HCl (pH 7.5) and 50 mM KCl in place of 30 mM potassium phosphate (pH 6.0). However, at pH 7.5, dnaB complementing activity could not be recovered in the absence of ATP in the presence or absence of dnaC(D) gene product. 921