Function of DNA Polymerase III in DNA Replication

Nüsslein, Volker; Otto, Bernd; Bonhoeffer, Friedrich; Schaller, Heinz

doi:10.1038/newbio234285a0

Cited by 110 publications

(40 citation statements)

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“…The specific roles of the three known DNA polymerases are not known. The finding that the only residual polymerase activity observed in HMS83 is DNA polymerase III lends support to the studies of Gefter et al (12) and Nuisslein et al (14). Clearly polymerase III is essential for DNA replication, but its role in initiation and 10,2 po/A-po/ B" T. …”

Section: Discussionsupporting

confidence: 75%

“…In a survey of mutants temperature-sensitive for DNA synthesis, dnat8, Gefter et al (12) found that strains with a mutation at the dnaE locus (13) contained a thermolabile polymerase III. Niisslein et al (14) have partially purified the dnaE gene product using an in vitro complementation assay for replication, and have shown that the purified fraction contains an activity that has all of the properties of polymerase III. Therefore, it seems that polymerase III is essential for DNA replication.…”

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confidence: 99%

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Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

Campbell

Soll

Richardson

1972

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

125

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A mutant of Escherichia coli deficient in DNA polymerase II has been isolated from E. coli polAl by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and assay of polymerase activity in extracts of survivors. The polAl mutation was suppressed during mutagenesis by introduction of the suppressor, su7 +, into the parental strain. The mutant, HMS83 polAl polBi, contains less than 0.5% of the normal levels of DNA polymerase II. The only polymerase activity detected in the mutant is DNA polymerase III. E. coli HMS83 grows normally at both 25 and 420, and supports the growth of bacteriophages T4, T7, lambda, OX174, 186, P2, and fd. The poiB mutation does not affect sensitivity to ultraviolet irradiation or recombination frequencies.Three distinct DNA polymerases have been found in extracts of Escherichia coli. The major activity, polymerase I, has been purified to homogeneity and studied extensively (1). In order to determine the role of polymerase I in vivQ, DeLucia and Cairns (2) isolated a mutant of E. coli, E. coli polAl, that has greatly reduced levels of polymerase I activity in extracts. The mutant grows normally, but has definite physiological defects in repair mechanisms (2, 3).The residual polymerase activity detectable in extracts of E. coli polAl has been resolved into two enzymes: polymerase II (4-11) and polymerase III (5). In a survey of mutants temperature-sensitive for DNA synthesis, dnat8, Gefter et al. (12) found that strains with a mutation at the dnaE locus (13) contained a thermolabile polymerase III. Niisslein et al. (14) have partially purified the dnaE gene product using an in vitro complementation assay for replication, and have shown that the purified fraction contains an activity that has all of the properties of polymerase III. Therefore, it seems that polymerase III is essential for DNA replication.In contrast to polymerase III, polymerase II activity is normal in all the known classes of dnat8 mutants (7,12). Thus, in order to investigate the function of polymerase II in vivo, we decided to isolate a mutant lacking the enzyme. We tried to minimize the bias inherent in any selection by screening a highly mutagenized stock of E. coli for polymerase II in individual extracts. Because DNA polymerase I activity masks polymerase II activity in extracts, it was necessary to use E. coti polAl as the parental strain. This communication describes the isolation and partial characterization of a mutant lacking DNA polymerase II in extracts. MATERIALS AND METHODSBacterial and Phage Strains. The mutants were isolated from strains derived from E. coli W3110 thy-. The parental strain was JG138 thy-polAl rha-tacZam. Bacteriophage P1KC was grown on LS448F'14 and used for the transduction to su7+. (Calbiochem,10,000 units/mg) was added. The tubes were incubated for 5 min at 370, then chilled to 00 after the addition of 0.1 ml of 25% Triton X-100. The 2090 Abbreviations: NTG, N-methyl-N'-nitro-N-nitrosoguanidine; MMS, methylmethanesulfonate.

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Section: Discussionsupporting

confidence: 75%

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confidence: 99%

Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

Campbell

Soll

Richardson

1972

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

125

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“…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). Of the other proteins known to be involved in E. coli DNA replication, dnaB, C(D), and G have been isolated using in vitro complementation assays in which protein preparations from wild-type cells stimulate 4X174 DNA-dependent DNA synthesis in heat-inactivated crude extracts of dna temperature-sensitive cells (16)(17)(18)(19).…”

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

Wickner¹,

Hurwitz²

1975

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

172

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(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

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“…An exponentially growing culture of E. coli B was incubated with 3H-NAL (total concentration, 5 ,ug/ml) for 15 min at 37 C. The culture was rapidly chilled to 0 C, made acidic by the addition of concentrated HCl, and then extracted with CHCl3. NAL is very soluble in CHCl3 and insoluble in aqueous solutions when the pH is less than 7. After separation of the two phases, the CHCI3 phase, containing about 98% of the input radioactivity, was concentrated to about 0.1 ml by evaporation and then chromatographed by thin-layer chromatography (TLC).…”

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Studies on the Mechanism of Action of Nalidixic Acid

Bourguignon

Levitt

Sternglanz

1973

Antimicrob Agents Chemother

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With three independent techniques (absorption spectrophotometry, measurement of the deoxyribonucleic acid [DNA] melting temperature, and equilibrium dialysis), no evidence has been found for the binding of nalidixic acid to purified DNA. Also, no evidence has been found to support the hypothesis that nalidixic acid is permanently modified to a new, active compound by the bacterial cell. By using an in vitro DNA replication system developed by Bonhoeffer and colleagues, soluble extracts from nalidixic acid-sensitive cells have been shown to confer nalidixic acid sensitivity on the DNA synthesis of lysates from nalidixic acid-resistant cells. The activity in the extracts is only present in sensitive cells and is nondialyzable and heat sensitive. Finally, two known nalidixic acid-resistant mutants of Escherichia coli, mapping at nal A and nal B, respectively, have been tested to determine whether either of them is a transport mutant. It has been shown that nal Br is a transport mutant whereas nal Ar is not.Nalidixic acid (NAL; 1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine -3 -carboxylic acid) is a specific, rapid, and reversible inhibitor of bacterial deoxyribonucleic acid (DNA) replication (3, 9). At present the mechanism of action of NAL is totally unknown. Previous work by other investigators has ruled out several possibilities. For example, none of the many purified enzymes involved in DNA metabolism are inhibited by NAL in vitro. Those (13,16,18). Consequently, NAL must be blocking some aspect of the DNA polymerization reaction itself and not just synthesis of the DNA precursors. Based on the work described above, three possibilities remain for the way in which NAL inhibits DNA replication: (i) NAL binds directly to the DNA template; (ii) NAL binds to and inactivates one of the components of the DNA replication complex (possibly an unknown replication protein); or (iii) NAL is chemically modified by metabolizing bacteria to an active form, which then functions by either (i) or (ii). In this paper we present experiments which test all three of these possribilities.In addition, we have carried out experiments with the two known NAL-resistant mutants of E. coli, mapping at nal A and nal B (10)

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Function of DNA Polymerase III in DNA Replication

Cited by 110 publications

References 1 publication

Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

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

Studies on the Mechanism of Action of Nalidixic Acid

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