On the molecular basis of the thermal sensitivity of an Escherichia coli topA mutant.

Studies of two temperature-sensitive Escherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity. We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17. We find that the supD allele in this strain had lost its temperature sensitivity. The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive. Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 degrees C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 degrees C, a nonpermissive temperature. Our results thus implicate an indispensable role of DNA topoisomerase I in E. coli cells grown in media of any osmolarity.     

Changed properties of the A subunit in DNA gyrase with a B subunit mutation

Summary To investigate the interaction of subunits A and B of DNA gyrase during DNA supercoiling, a Cou^r mutant of Escherichia coli was obtained and the effect of nalidixic acid on the supercoiling of DNA by wild-type and mutant enzymes was assayed. The enzyme of the Cou^r strain proved to be more sensitive to nalidixic acid than the wild-type DNA gyrase. Hence the mutation affecting the B subunit can also change the properties of the A subunit, which fact suggests that the two subunits of DNA gyrase are in contact during DNA supercoiling.     

Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid.

A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.     

Immunoaffinity-purified DNA polymerase alpha from a mouse temperature-sensitive mutant, tsFT20 strain, is heat-labile

We have purified DNA polymerase alpha from a temperature-sensitive mutant cell line of mouse FM3A cells, tsFT20, that shows temperature-sensitive activity of DNA polymerase alpha (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1761-1765). The purified enzyme was composed of two polypeptides with the same apparent molecular weights as those of purified DNA polymerase alpha from the parental strain, FM3A (Mr 180,000 and 68,000). Heat inactivation experiments revealed that this purified DNA polymerase alpha from tsFT20 cells was more heat-labile than the wild-type enzyme. We have also purified primase from both ts-FT20 cells and wild-type cells. Both primase fractions consist of two polypeptides with molecular weights of 54,000 and 46,000. No difference was observed between the heat labilities of the primases from tsFT20 cells and wild-type cells. Comparisons of wild-type and mutant polymerase indicated that the temperature-sensitive mutation in DNA polymerase alpha from tsFT20 cells affect the dCTP-binding site of the enzyme. The mutation also changed the optimum pH and the optimum KCl concentration of the enzyme.     

Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis

Insertion and deletion mutagenesis within the gene topA of Escherichia coli encoding DNA topoisomerase I was carried out to test the existence of subdomains in the enzyme and the relationship between the slow-growth topA- phenotype and the known DNA relaxation activity of the enzyme. All mutants that show no detectable DNA relaxation activity in cell extracts fail to complement the temperature-sensitive growth defect of strain AS17 topAam harboring a plasmid-borne temperature-sensitive suppressor tRNA. All mutants that show partial or full levels of DNA relaxation activity in cell extracts (relative to activity in extracts of wild-type cells) can complement this defect. The carboxyl-proximal 25% of the enzyme appears to be in a domain that is dispensable both in terms of the catalytic function of the enzyme and its biological role. Analysis of the mutant enzyme also indicates that the formation of the covalent topoisomerase-DNA complex is correlated with the DNA relaxation activity, which supports the notion that the covalent complex is an obligatory intermediate in the catalysis of DNA topoisomerization.     

Restriction fragment length polymorphism of polymerase chain reaction products from vaccine and wild-type varicella-zoster virus isolates.

The nucleotide changes that result in two restriction endonuclease polymorphisms that differentiate wild-type varicella-zoster virus (VZV) from the vaccine strain were determined. Oligonucleotide primers that flank these sites were used to amplify the intervening sequences with the polymerase chain reaction to identify VZV DNA in clinical isolates. Restriction enzyme digestion of the amplification products distinguished vaccine and wild-type genomes from one another. This study confirms the feasibility of amplifying VZV sequences so that they may be detected in clinical specimens and provides a molecular epidemiological approach to strain identification of VZV in vesicular lesions.     

Role of uracil-DNA glycosylase in the repair of deaminated cytosine residues of DNA in Escherichia coli.

Uracil-DNA glycosylase, which acts specifically on uracil-containing DNA, was purified 250-fold from an extract of Escherichia coli 1100. The enzyme releases free uracil from DNA, producing alkali-labile apyrimidinic sites in the DNA. The enzyme is active on both native and heat-denatured DNA of phage PBS1, which contains uracil in place of thymine. piX174 DNA which had been treated with bisulfite and then at alkaline pH was susceptible to the action of uracil-DNA glycosylase. Since DNA treated with bisulfite alone was less susceptible to the enzyme, it is likely that the enzyme recognizes deaminated cytosine, namely uracil, but not bisulfite adducts of uracil and cytosine in the treated DNA. DNA treated with nitrite or hydroxylamine was not attacked by the enzyme. Enzyme activity acting on bisulfite-treated DNA was absent from an extract of E. coli mutant BD10 (ung). The mutant exhibited higher sensitivity to bisulfite than did the wild-type strain and was unable to reactivate phage T1 pre-exposed to bisulfite and weak alkali.     

Morganella morganii urease: purification, characterization, and isolation of gene sequences

Morganella morganii, a very common cause of catheter-associated bacteriuria, was previously classified with the genus Proteus on the basis of urease production. M. morganii constitutively synthesizes a urease distinct from that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular size of 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6 kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree of conservation of amino acid sequence between jack bean and Proteus mirabilis ureases. Km for urea equalled 0.8 mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h of incubation. All urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did not precipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunits of ureases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmid clones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequences were localized to a 7.1-kb EcoRI-SalI fragment. Tn5 mutagenesis revealed that between 3.3 and 6.6 kb of DNA were necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences of other species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blots of cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded to those seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amounts of urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.     

Compromised cellular responses to DNA damage accelerate chronological aging by incurring cell wall fragility in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Elevated levels of reactive oxygen species (ROS) can attack almost all cell components including genomic DNA to induce many types of DNA damage. In this study, we used Saccharomyces cerevisiae with various mutations in a biological network supposed to prevent deleterious effects of endogenous ROS to test the effect of such a network on yeast chronological aging. Our results showed that cells with defects in cellular antioxidation, DNA repair and DNA damage checkpoints displayed a mutation rate higher than that of wild-type strain. Moreover, the chronological life span of most mutants as determined by colony formation was found to be shorter than that of wild-type cells, especially for the mutants defective in DNA replication and DNA damage checkpoints, although the observed cell number was almost the same for wild-type and mutant strains. The mutants were finally found to be more sensitive to SDS and lysing enzyme treatment, and that the degree of sensitivity was correlated with their chronological life span.     

Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay.

Two mutations affecting herpes simplex virus type 1 glycoprotein B were mapped by marker rescue using cloned sequences of wild-type herpes simplex virus type 1 strain KOS DNA. One mutant, tsB5, is a temperature-sensitive mutant which does not express mature, functional glycoprotein B at the nonpermissive temperature. The other mutant, marB1.1, expresses an antigenic variant of glycoprotein B and was selected for resistance to neutralization by a monoclonal antibody. The mutation in tsB5 mapped to a 1.2-kilobase segment of the herpes simplex virus type 1 genome between coordinates 0.361 and 0.368, whereas the mutation in marB1.1 mapped to a 1.6-kilobase segment between coordinates 0.350 and 0.361. An in situ enzyme immunoassay was used to detect plaques of recombinant wild-type virus among the progeny of transfections with mutant marB1.1 DNA and wild-type DNA fragments.     

Studies of propionate toxicity in Salmonella enterica identify 2-methylcitrate as a potent inhibitor of cell growth

Salmonella enterica serovar Typhimurium LT2 showed increased sensitivity to propionate when the 2-methylcitric acid cycle was blocked. A derivative of a prpC mutant (which lacked 2-methylcitrate synthase activity) resistant to propionate was isolated, and the mutation responsible for the newly acquired resistance to propionate was mapped to the citrate synthase (gltA) gene. These results suggested that citrate synthase activity was the source of the increased sensitivity to propionate observed in the absence of the 2-methylcitric acid cycle. DNA sequencing of the wild-type and mutant gltA alleles revealed that the ATG start codon of the wild-type gene was converted to the rare GTG start codon in the revertant strain. This result suggested that lower levels of this enzyme were present in the mutant. Consistent with this change, cell-free extracts of the propionate-resistant strain contained 12-fold less citrate synthase activity. This was interpreted to mean that, in the wild-type strain, high levels of citrate synthase activity were the source of a toxic metabolite. In vitro experiments performed with homogeneous citrate synthase enzyme indicated that this enzyme was capable of synthesizing 2-methylcitrate from propionyl-CoA and oxaloacetate. This result lent further support to the in vivo data, which suggested that citrate synthase was the source of a toxic metabolite.     

Deoxyribonucleic Acid Repair in a Highly Radiation-Resistant Strain of Salmonella typhimurium

Deoxyribonucleic acid repair was studied in gamma-irradiated wild-type Salmonella typhimurium and in a radiation-resistant derivative 20 times more resistant than wild type. After exposure to 20 or 50 krad, the wild-type strain (DB21) degraded 30 to 50% of its prelabeled DNA into acid-soluble fragments, whereas the radioresistant strain degraded less than 15% after 4 h of incubation. Post-irradiation synthesis of DNA in the wild-type strain DB21 was reduced after a dose of 20 krad and totally inhibited after exposure to 200 krad. With radiation-resistant strain, D21R6008, on the other hand, DNA synthesis was delayed after a dose of 200 krad but not inhibited. Doses of 20 and 200 krad produced a similar number of single-strand breaks in the DNA of both strains as determined by zone sedimentation analysis in alkaline sucrose gradients. The radiation-resistant strain D21R6008, on the other hand, DNA synthesis was strand breaks in its DNA and repairs these damages more rapidly than wild-type Salmonella.     

Repair of cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium and Escherichia coli.

The role of nucleotide excision repair and 3-methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3-methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.     

Cloning of the gene for Escherichia coli glutamyl-tRNA synthetase

The structural gene for the glutamyl-tRNA synthetase of Escherichia coli has been cloned in E. coli strain JP1449, a thermosensitive mutant altered in this enzyme. Ampicillin-resistant and tetracycline-sensitive thermoresistant colonies were selected following the transformation of JP1449 by a bank of hybrid plasmids containing fragments from a partial Sau3A digest of chromosomal DNA inserted into the BamHI site of pBR322. One of the selected clones, HS7611, has a level of glutamyl-tRNA synthetase activity more than 20 times higher than that of a wild-type strain. The overproduced enzyme has the same molecular weight and is as thermostable as that of a wild-type strain, indicating that the complete structural gene is present in the insert. These characteristics were lost by curing this clone of its plasmid with acridine orange, and were transferred with high efficiency to the mutant strain JP1449 by transformation with the purified plasmid. A physical map of the plasmid, which contains an insert of about 2.7 kb in length, is presented.     

A mutant of Escherichia coli K12 deficient in the 5''-3'' exonucleolytic activity of DNA polymerase I. II. Purification and properties of the mutant enzyme

Summary The enzymatic properties of purified DNA polymerase I from a strain of Escherichia coli K12 with a mutation in the polA gene have been studied. The polymerizing activity of the mutant enzyme is similar to that of the enzyme from isogenic wild-type cells, when the activity is measured on exonuclease III treated calf-thymus DNA. Also the 3–5 exonucleolytic activity is not significantly different for both enzyme preparations. The 5–3 exonucleolytic activity of DNA polymerase I isolated from the mutant strain, however, is much lower than that of wild-type DNA polymerase I. The products formed by the action of the wild-type and the mutant enzyme on nicked circular double-stranded DNA of phage X174 (RFII DNA) were analysed by sucrose-gradient sedimentation and electron-microscopy. When RFII DNA was incubated with wild-type enzyme 80% of the molecules were converted into linear molecules. All linear molecules were shorter than one phage genome. Only 25% of the molecules were branched. After incubation of RFII DNA with the mutant enzyme 62% of the molecules have become linear. More than 90% of these linear molecules were branched and the majority of them was longer than one phage genome.     

Functions of multiple exonucleases are essential for cell viability, DNA repair and homologous recombination in recD mutants of Escherichia coli

Heterotrimeric RecBCD enzyme unwinds and resects a DNA duplex containing blunt double-stranded ends and directs loading of the strand-exchange protein RecA onto the unwound 3'-ending strand, thereby initiating the majority of recombination in wild-type Escherichia coli. When the enzyme lacks its RecD subunit, the resulting RecBC enzyme, active in recD mutants, is recombination proficient although it has only helicase and RecA loading activity and is not a nuclease. However, E. coli encodes for several other exonucleases that digest double-stranded and single-stranded DNA and thus might act in consort with the RecBC enzyme to efficiently promote recombination reactions. To test this hypothesis, I inactivated multiple exonucleases (i.e., exonuclease I, exonuclease X, exonuclease VII, RecJ, and SbcCD) in recD derivatives of the wild-type and nuclease-deficient recB1067 strain and assessed the ability of the resultant mutants to maintain cell viability and to promote DNA repair and homologous recombination. A complex pattern of overlapping and sometimes competing activities of multiple exonucleases in recD mutants was thus revealed. These exonucleases were shown to be essential for cell viability, DNA repair (of UV- and gamma-induced lesions), and homologous recombination (during Hfr conjugation and P1 transduction), which are dependent on the RecBC enzyme. A model for donor DNA processing in recD transconjugants and transductants was proposed.     

A mutation in the consensus ATP-binding sequence of the RecD subunit reduces the processivity of the RecBCD enzyme from Escherichia coli.

We have constructed a mutant form of the RecBCD enzyme from Escherichia coli with a lysine to glutamine change in the consensus ATP-binding sequence in the RecD subunit (Korangy, F., and Julin, D.A. (1992a, 1992b) J. Biol. Chem., 1727-1732; 1733-1740). We compare here the kinetics of double-stranded DNA-dependent ATP hydrolysis by the mutant (RecBCD-K177Q) and wild-type enzymes. We included heparin to trap enzyme not bound to DNA, or the single-stranded DNA-binding (SSB) protein from Escherichia coli to prevent the enzyme from binding to single-stranded DNA products and partially single-stranded reaction intermediates. The ATP hydrolysis kinetics in either case show a rapid burst phase followed by a slower second phase. The wild-type enzyme hydrolyzes an amount of ATP about equal to the DNA nucleotide concentration in the rapid phase. The amount of ATP hydrolyzed by the RecBCD-K177Q enzyme in the burst is about 8-10-fold lower than the wild-type, in the presence of either heparin or SSB. The burst magnitude of the wild-type enzyme with heparin is proportional to the size of the DNA from about 1,420 to 22,400 base pairs whereas that of the mutant is independent of the DNA size. The wild-type enzyme completely degrades a 6,250-base pair DNA substrate with no partially degraded molecules visible on agarose gels. RecBCD-K177Q enzyme reaction mixtures in the presence of SSB protein contain a heterogeneous mixture of partially degraded molecules of 2,000-5,000 base pairs. These results indicate that the RecBCD-K177Q enzyme is less processive than the wild-type enzyme.     

Interactions of subunits of DNA gyrase

Interaction of DNA gyrase A- and B-subunits during the process of DNA supercoiling was studied. For this purpose a E. coli Cour-1 mutant resistant to coumermycin and containing a mutation in the B-subunit of DNA gyrase was isolated and the influence of the DNA gyrase A-subunit specific inhibitor-nalidixic acid-on DNA supercoiling by wild-type and mutant enzymes was investigated. It turned out that the enzyme from the Cour-1 mutant strain was more sensitive to nalidixic acid than the DNA gyrase from the wild-type strain. Hence, the mutation affecting the B-subunit is capable to change A-subunit properties. That makes it possible to draw the conclusion about a close structural interaction of DNA gyrase subunits during DNA supercoiling.