A Mutant of
            <i>Escherichia coli</i>
            Defective in DNA Polymerase II Activity

Hirota, Yukinori; Gefter, Malcolm L.; Mindich, Leonard

doi:10.1073/pnas.69.11.3238

Cited by 64 publications

(30 citation statements)

References 17 publications

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“…The function of ftsM in Weigle reactivation and its regulation of lexA (12) suggest a role in SOS-associated repair. The map position of the ftsM gene near leu (12) does not exclude the possibility that ftsM is an allele of polB (7,16) and dinA (20).…”

Section: Discussionmentioning

confidence: 97%

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments

et al. 1988

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Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments. To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM). Already at the permissive temperature, all mutants, particularly the pbpB and ftsQ mutants,showed an increased average cell length and cell mass. The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period. All strains, except ftsZ, continued to initiate new constrictions at 42rC, suggesting the involvement of FtsZ in an early step of the constriction process. The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions. Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly. Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times L0, the length of newborn cells at the permissive temperature. The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells. Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3. TheftsE mutant continued to constrict and divide at 42°C, forming short filaments, which recovered quickly after a shift back to the permissive temperature. After prolonged growth at 42°C, chains of cells, which eventually swelled up, were formed. Although theftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions. Escherichia coli is one of the very few organisms in which the genetics of the regulation of cell division can be studied: a number of specific conditional mutants that are affected in cell division have been described (for a review, see reference 11), and an enzymatic activity of one of the gene products has been demonstrated (18). Cell division in E. coli occurs by a seemingly simple ingrowth of the envelope layers without the help of additional structures like external wall bands (15) or internal skeletal compounds (30). In spite of this simplicity, it has appeared difficult to determine the specific role of the various cell division genes. First, the composition of the envelope layers at the division site has not yet been shown to differ chemically from the rest of the envelope. As a result, we have to describe the process in morphological terms and distinguish visual stages like initiation of cell wall ingrowth, formation of polar caps, and cell separation. The second reason is that inhibition of cell division can be the result of even slight perturbations of DNA synthesis (the SOS response), protein synthes...

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Section: Discussionmentioning

confidence: 97%

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments

et al. 1988

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“…Initial biochemical studies of DNA Pol II (13,22,36) did not provide any major insights as to its possible role, and mutants defective in the enzyme (polB) (7,17) showed no discernible phenotype. The gene encoding Pol II has been cloned and sequenced previously (la, 8, 9, 19), and mutants totally lacking Pol II have been isolated, thus establishing that Pol II is not required for cell viability (1).…”

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confidence: 99%

Absence of a role for DNA polymerase II in SOS-induced translesion bypass of phi X174

et al. 1993

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In order to examine the possible role of Escherichia coli DNA polymerase H in SOS-induced translesion bypass, Weigle reactivation and mutation induction were measured with single-stranded 4X174 transfecting DNA containing individual lesions. No decrease in bypass of thymine glycol or cyclobutane pyrimidine dimers in the absence of DNA polymerase H was observed. Furthermore, DNA polymerase H did not affect bypass of abasic sites when either survival or mutagenesis was the endpoint. Lastly, repair of gapped DNA molecules, intermediates in methyl-directed mismatch repair, was also unaffected by the presence or absence of DNA polymerase II.The cellular function of DNA polymerase II (Pol II) of Escherichia coli has eluded biochemists and geneticists since it was originally identified over 20 years ago (20,21,28 (la, 19). Taken together, these data suggest that DNA Pol II is involved in cellular DNA repair.It has been commonly thought and most data have suggested that the DNA polymerase involved in lesion bypass and mutagenesis is the DNA Pol III holoenzyme (4, 5, 14), and genetic studies have shown that RecA, UmuC, and UmuD are required for mutagenesis produced by UV lesions (33) and abasic sites (26). Weigle reactivation (35), an increase in survival of damaged phage or phage DNA, also requires these gene products (6,11,12 DNA containing DNA damage. Weigle reactivation of singlestranded and duplex DNA phages and transfecting DNA has been used as a measure of lesion bypass in a number of systems. It is a sensitive and simple method, since increases or decreases in survival are quite easily scored. We elected to examine the potential role of Pol II in the survival of two lesions, thymine glycol and the cyclobutane pyrimidine dimer, that are strong blocks to DNA polymerases in uninduced cells but are efficiently bypassed in SOS-induced cells. Thymine glycol, an oxidative DNA damage that is lethal in uninduced cells, is the most efficiently bypassed lesion examined to date in induced cells (34). A total of 60 to 70% of thymine glycols are bypassed in both single-stranded (16) and duplex (25) DNAs, whereas 30 to 40% of cyclobutane pyrimidine dimers are bypassed (38). For both lesions, we used single-stranded damage-containing DNA to remove any possible effects of excision and recombination repair processing from the endpoint. Figure 1 shows the survival response of damaged DNA containing either thymine glycols (Fig. 1A) or cyclobutane pyrimidine dimers (Fig. 1B) with increasing damage-inducing UV light dose to the host. As can be seen in both cases, Weigle reactivation was substantial, but no difference was observed whether the strain contained or lacked DNA PolII. Furthermore, the results shown here for the dinA Mud(Apr lac) fusion were also obtained with a polB point mutant, a polB deletion (SH2101; unpublished observations), or a double mutant defective in both DNA PolI and Pol II (poLA polB; data not shown). Thus, DNA Pol II does not appear to be involved in Weigle reactivation of readily bypassable lesions.We and o...

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“…Therefore, the map position of ispA on the E. coli chromosome was determined. Since the phenotype of the ispA mutant is not easily distinguishable from that of the ispA+ strain, the ispA mutation of JE11046 was mapped by the indirect mapping technique described previously (15). ispA mutant JE11046 was crossed with strain P4X8, and the selection was made on one or several markers carried by JE11046.…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase

et al. 1989

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The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-ion. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30°C and 5% of that at 40°C. At 42°C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30°C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30°C and almost the same in both strains at 42°C. The mutant had no obvious phenotype regarding its growth properties.It is well known that 3-hydroxy-3-methylglutaryl coenzyme A reductase is a rate-limiting enzyme in the biosyntheses of isoprenoid compounds, undergoing multivalentfeedback regulation (4). However, a branch point of the synthetic pathway of various isoprenoids seems to be a reaction utilizing isopentenyl diphosphate (IPP) or farnesyl diphosphate (FPP) or both. For elucidation of the mechanism by which the syntheses of various isoprenoids are regulated differently, many researchers have studied the regulation of activities of the enzymes, utilizing IPP or FPP or both as a substrate (1,(5)(6)(7)11).Escherichia coli includes isoprenoid quinones (ubiquinone-8, menaquinone-8, and demethylmenaquinone-8) having an all-E-octaprenyl side chain (8) and sugar carrier lipid (decaprenyl phosphate, undecaprenyl phosphate, and dodecaprenyl phosphate) containing a Z,E-mixed isoprenoid chain (27). Because the composition of isoprenoids in E. coli is simpler than that in eucaryotes, E. coli is a useful system as a model for studying the biosynthesis of nonsterol isoprenoids and its regulation. We have studied the biosynthetic enzymes, utilizing IPP as a substrate in E. coli, and separated IPP isomerase and three prenyltransferases, namely, FPP synthase, octaprenyl diphosphate synthase, and undecaprenyl diphosphate synthase, from each other (13). FPP synthase catalyzes the condensation of IPP with dimethylallyl diphosphate (C5) or geranyl diphosphate (C1O) to give FPP (C15). The latter two enzymes catalyze the sequential condensation of IPP with FPP to give long-chain polyprenyl diphosphates which are precursors of isoprenoid quinones and sugar carrier lipid.One effective approach to the elucidation of the role of a certain enzyme in vivo is to isolate the mutant containing the defective enzyme and to characterize it. We screened a collection of temperature-sensitive mutants of E. coli (24) for defects in isoprenoid synthesis by measuring 14C incorpora-* Corresponding author. Screening of temperature-sensitive mut...

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A Mutant of Escherichia coli Defective in DNA Polymerase II Activity

Cited by 64 publications

References 17 publications

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments

Absence of a role for DNA polymerase II in SOS-induced translesion bypass of phi X174

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase

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