Isolation and Partial Characterization of a Mutant of
            <i>Escherichia coli</i>
            Deficient in DNA Polymerase II

A mutant of E. coli defective in DNA polymerase II activity was isolated. Extracts had about 0.1% of the normal activity of the DNA polymerase. The effect of the mutation on the ability of cells to replicate DNA of various sources was analyzed. Mutant bacteria grow normally, at 410 as well as at 300. All bacteriophages, F factors, and R factors examined so far grow normally in the mutant. Sensitivity of the mutant to ultraviolet radiation and alkylating reagents in growth media was the same as that of the wild type.The mutation, polBI, is recessive with respect to wild type. The polBI mutation can coexist with recombinationdefective mutations. Genetic mapping studies show the mutation to be located at about 2 min on the E. coli map.Isolation of an amber mutant of E. coli, polAl (1), which is defective in the DNA polymerase described by Kornberg (2) (DNA polymerase I), yet grows normally, led to the conclusion that DNA polymerase I might not be a necessary component for replication of E. coli DNA. It has been suggested that this enzyme plays a role in DNA repair (1) and in replication of a colicinogenic factor El (3, 4). Availability of this mutant stimulated enzymological investigations for new DNA polymerases: DNA polymerase II (5-8) and DNA polymerase III (5) were discovered, purified, and characterized.We have analyzed these DNA polymerase activities in a series of thermosensitive mutants defective in DNA replication, dnaA through G. Normal levels of activity were found for DNA polymerase I and II; however, strains having thermosensitive mutations at the dnaE locus were defective in DNA polymerase III (9). We concluded, therefore, that DNA polymerase III is an enzyme required for DNA replication in E. coli.In this paper, we report the isolation and characterization of a mutant of E. coli defective in DNA polymerase II activity. MATERIALS (11) 5, 103, 145, 160, 193, 218, 219, 266, 326, and 328, were the kind gift of Dr. G. Bertani.Media. L-broth and L-agar (12) supplemented with 50 /Ag of thymine per ml and 2.5 mM CaCl2 were used to grow bacterial cultures and for assays of phage and bacteria.Minimal synthetic medium 63 (13) supplemented with 0.2% glucose and 5 /Ag of thymine per ml, was used. Synthetic medium with 0.2% glucose and 5 ,ug of thymine per ml was used. Synthetic medium 63 supplemented with 50 1Ag of novobiocin per ml, a gift of Laboratories Upjohn (Paris), and McConkey-medium (Difco Laboratories) were used to score a mutation defective in lipopolysaccharide synthesis (14).Bacterial Crosses. Bacteria, Hfr and F-, exponentially growing at 300 in broth supplemented with thymine were mixed (1 Hfr/10 F-) in the same medium. After 2 hr at 300, the cultures were washed, diluted, and plated on appropriate media. The plates was incubated at 300. Recombinant colonies were isolated from the plates, purified, and tested for their character by replica plating on suitable media. Enzyme Assay. Procedures used for growth of cells, preparation of cell-free extracts, and separation of polymerases II an...

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“…A similar E. coli mutant has also been isolated and characterized by others (24), and the results are generally in agreement with ours.…”

Section: Mapping Of Polbi and Dnaesupporting

confidence: 81%

A Mutant of Escherichia coli Defective in DNA Polymerase II Activity

Hirota

Gefter

Mindich³

1972

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

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“…Normal levels of Pol II were found for strain HMS83 (Fig. 1, lane 3), an E. coli mutant shown previously to be devoid of DNA Pol II enzyme activity, using either a standard polymerase incorporation assay (8) or an in situ polyacrylamide gel assay (4). Pol II cross-reacting material was absent in strains MC1061 (Fig.…”

Section: Materuils and Methodsmentioning

confidence: 99%

Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation

et al. 1994

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DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity ( (31,34,35,45).Recently, the gene encoding DNA polymerase II has been cloned (3,11,27), and its structural gene was identified as the SOS damage-inducible dinA gene (3,27). Pol II is part of the SOS regulon, having a LexA repressor operator site present in the promoter of the gene (3,27). Earlier, we had shown that Pol II could bypass a single site-directed abasic lesion in vitro, and levels of the enzyme in vivo exhibited a sevenfold increase

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“…However, no biological role has been assigned to DNA polymerase II, encoded by polB. The polB mutants that have been isolated exhibit no defect in DNA replication and DNA repair (5,9).…”

mentioning

confidence: 99%

The Escherichia coli polB gene, which encodes DNA polymerase II, is regulated by the SOS system

et al. 1990

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The dinA (damage inducible) gene was previously identified as one of the SOS genes with no known function; it was mapped near the leuB gene, where the poiB gene encoding DNA polymerase H was also mapped. We cloned the chromosomal fragment carrying the dinA region from the ordered Escherichia coli genomic library and mapped the dinA promoter precisely on the physical map of the chromosome. The cells that harbored multicopy plasmids with the dimA region expressed very high levels of DNA polymerase activity, which was sensitive to N-ethylmaleimide, an inhibitor of DNA polymerase II. Expression of the polymerase activity encoded by the dinA locus was regulated by SOS system, and the dinA promoter was the promoter of the gene encoding the DNA polymerase. From these data we conclude that the polB gene is identical to the dinA gene and is regulated by the SOS system. The product of the polB (dinA) gene was identified as an 80-kDa protein by the maxicell method.In Escherichia coli, three DNA polymerases have been identified (15). DNA polymerase III has been shown to be essential for chromosomal DNA replication and to consist of multiple species of subunits. The nucleotide polymerizing activity resides in the a subunit encoded by the dnaE (poIC) gene. DNA polymerase I, encoded by polA, has been shown to be involved in DNA repair and to be required for DNA replication when the cell is growing fast on rich medium (11). However, no biological role has been assigned to DNA polymerase II, encoded by polB. The polB mutants that have been isolated exhibit no defect in DNA replication and DNA repair (5, 9).We suspected that the existing polB mutants retained substantial levels of DNA polymerase II activity in vivo and that this might have prevented us from revealing the specific phenotype of the mutants so far. To elucidate the role of DNA polymerase II in DNA replication, repair, recombination, and mutagenesis, we thought it necessary to construct apolB deletion mutant by using a cloned gene (25). Biochemical studies on DNA polymerase II have been hampered by the relative scarcity of the enzyme in the cell. The overproduction of the enzyme with the cloned gene would facilitate these studies. Therefore, we have been trying to clone the polB gene. Bonnar et al. (3) presented evidence that synthesis of DNA polymerase X, presumably DNA polymerase II, is regulated by LexA repressor. The dinA gene (13) was identified as an SOS-regulated gene with no known function; it was mapped near leuB, where polB was also mapped (4, 9). It occurred to us that dinA might be identical to polB. In this work, we examined this possibility and found that it is indeed so. The polB gene was recently cloned by Chen et al. (6).MATERIALS AND METHODS Bacterial strains, plasmids, phages, and media. The E. coli strains used in this study are listed in Table 1. Strain HRS5200 was constructed by P1 transduction of leu+ and polB1OO of HMS83 (5) cysteine, and 14C-labeled proteins for molecular weight standards were purchased from Amersham Japan (Tokyo, Japan). M...

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

Cited by 125 publications

References 29 publications

A Mutant of Escherichia coli Defective in DNA Polymerase II Activity

A Mutant of Escherichia coli Defective in DNA Polymerase II Activity

Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation

The Escherichia coli polB gene, which encodes DNA polymerase II, is regulated by the SOS system

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