DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.     

Isolation and characterization of beta-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar Typhimurium

FabG, beta-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The beta-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.     

Role of the simian virus 40 gene A product in regulation of DNA synthesis in transformed cells.

Cells transformed by tsA mutants of simian virus 40 (SV40) are temperature sensitive for the maintenance of the transformed phenotype. The kinetics of induction of DNA synthesis were determined for hamster cell transformants shifted to the permissive temperature after a 48-h serum arrest at the nonpermissive temperature. DNAsynthesis was initiated in the tsA transformants by 8 h after shiftdown was maximal by 12 h. The presence or absence of fetal bovine serum at the time of temperature shift had no effect on the kinetics of initiation of DNA synthesis. Analysis of TTP in tsA transformants revealed similar levels of incorporation of [3H]thymidine into TTP at both permissive and nonpermissive temperatures. Autoradiography revealed that by 12 h after a shift to the permissive temperature, approximately 50% of the cells exhibited labeled nuclei after a 60-min pulse with [3H]thymidine, indicating that a majority of the cells were actively synthesizing DNA. By 8 to 12 h after a shiftup of confluent tsA transformants to the nonpermissive temperature, the number of labeled nuclei was reduced to approximately 16%, regardless of serum concentration. These data indicate that the SV40 gene A product, either directly or indirectly, regulates cellular DNA synthesis in transformed cells.     

Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

Thermolabile in vivo DNA-binding activity associated with a protein encoded by mutants of herpes simplex virus type 1.

The major DNA-binding protein encoded by several temperature-sensitive mutants of herpes simplex virus type 1 was thermolabile for binding to intracellular viral DNA. The ability of DNase I to release this protein from isolated nuclei was used as a measure of the amount of protein bound to viral DNA. This assay was based upon our previous observation that the fraction of herpesviral DNA-binding protein which can be eluted from nuclei with DNase I represents proteins associated with progeny viral DNA (D. M. Knipe and A. E. Spang, J. Virol. 43:314-324, 1982). In this study, we found that several temperature-sensitive mutants encoded proteins which rapidly chased from a DNase I-sensitive to a DNase I-resistant nuclear form upon shift to the nonpermissive temperature. We interpret this change in DNase I sensitivity to represent the denaturation of the DNA-binding site at the nonpermissive temperature and the association with the nuclear framework via a second site on the protein. The DNA-binding activity measured by the DNase I sensitivity assay represents an important function of the protein in viral replication because three of five mutants tested were thermolabile for this activity. A fourth mutant encoded a protein which did not associate with the nucleus at the nonpermissive temperature and therefore would not be available for DNA binding in the nucleus. We also present supportive evidence for the binding of the wild-type protein to intracellular viral DNA by showing that a monoclonal antibody coprecipitated virus-specific DNA sequences with the major DNA-binding protein.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type2.

Fourteen temperature-sensitive mutants of human adenovirus type2, which differed in their plaquing efficiencies at at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts206 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a high-molecular-weight form of viral DNA.     

Temperature-sensitive mutants of Saccharomyces cerevisiae variable in the methionine content of their protein.

Temperature-sensitive mutants were derived from Saccharomyces cerevisiae Y5alpha by ethyl methane sulfonate mutagenesis, in a search for mutants that would produce methionine-rich protein at the nonpermissive temperature. A total of 132 mutant strains were selected which showed adequate growth on minimal medium at 25 degrees C but little or no growth on the same medium supplemented with a high concentration (2 mg/ml) of l-methionine at 37 degrees C. Several of these mutants were found to increase the proportion of methionine in their protein to much higher levels than that of the wild-type parent after a temperature shift from 25 to 37 degrees C. Two strains, 476 and 438, which were temperature sensitive only in the presence of methionine, produced cellular protein with methionine contents as high as 3.6 and 4.3%, respectively, when incubated in the presence of methionine. The former strain contained 2.5% methionine even when incubated at 37 degrees C in the absence of methionine. Wild strain Y5alpha, on the other hand, had 1.75% methionine under all conditions tested. Most temperature-sensitive mutants isolated had the same methionine content as the wild strain. It is concluded that the proportion of a specific amino acid, such as methionine, in S. cerevisiae protein can be altered by culturing certain temperature-sensitive mutants at an elevated temperature.     

Early changes in the synthesis of proteins with affinity for single-stranded DNA during the onset of transformation in NRK cells.

Early alterations in the synthesis of proteins which bind to single-stranded DNA have been examined following the onset of transformation in NRK cells transformed by a heat-sensitive mutant (ts339) of Rous sarcoma virus. Transformation was initiated by shifting quiescent cultures from nonpermissive to permissive temperatures. Cultures were prelabelled with [3H]leucine for several generations at the non-permissive temperature, and with [35S]methionine at times after shift to the permissive temperature. Cytosol extracts were passed through sequential columns of double-stranded and single-stranded DNA bound to cellulose. Within the first hour of transformation there was an increase in the synthetic rate of proteins binding tightly to single-stranded DNA, but not to double-stranded DNA. More loosely bound protein fractions showed no such early synthetic increase. Electrophoresis of the fraction eluted from single stranded DNA-cellulose with 2 M NaCl demonstrated the presence of a major protein of 93 000 daltons, which comprised more than 0.1% of the cytosol protein. The synthesis of the 93 000 dalton protein increased continuously over the first 4 h interval after the onset of transformation. The synthetic rate of a 35 000 dalton protein, a major DNA-binding polypeptide found in mammalian cells, began to increase after a 1-h lag, following the onset of transformation. The protein fraction containing the 93 000 dalton protein had considerable unwinding activity, depressing the melting temperature of poly(dA-dT) by 39 degrees C. The protein fraction containing the bulk of the 35 000 dalton protein did not have unwinding activity. Transformation-induced DNA synthesis was measured in cells made permeable to deoxyribonucleoside triphosphates at times after shift to the permissive temperature. It was determined that synthesis of DNA began within the first 1--2 h after the onset of transformation. We conclude that the early transformation-associated synthesis of SS93 and perhaps other proteins binding to single-stranded DNA may be related to early transformation-associated changes preparatory to DNA replication and subsequent growth.     

The bacteriophage P4 alpha gene is the structural gene for bacteriophage P4-induced RNA polymerase

Two temperature-sensitive mutants of satellite phage P4 which do not synthesize P4 DNA at the nonpermissive temperature have been isolated. One of these phage is mutated in the P4 alpha gene. It complements a P4 delta mutant, but not a P4 alpha amber mutant; both mutants are phenotypically identical to alpha amber mutants in all properties studied. They synthesize P4 early proteins 1 and 2 as well as two additional P4-induced early proteins, 5 and 6, which are described here. P4 late proteins are not synthesized by these mutants and cannot be transactivated by helper phage P2. The mutants are unable to transactivate P2 late proteins from a P2 AB mutant. The P4 RNA polymerase activity which has been suggested to be involved in P4 DNA synthesis is not detected at the nonpermissive temperature. The P4 polymerase activity in partially purified extracts prepared from cells infected with the mutant at the permissive temperature is temperature sensitive. Reduced activity is found in vitro when these extracts are preincubated at 41 degrees C or assayed at temperatures higher than 37 degrees C. Thus, the P4 RNA polymerase is the product of the alpha gene. Temperature shift experiments show that the alpha gene product is required until late in the P4 cycle.     

Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae.

cdc9, a temperature-sensitive mutant defective in polynucleotide deoxyribonucleic acid (DNA) ligase activity, accumulates low-molecular-weight DNA fragments (as measured by sedimentation of DNA in alkaline sucrose gradients) at the nonpermissive temperature after irradiation with ultraviolet light. This phenotype of cdc9 is a sensitive indicator of successful incision during excision repair of dimers. In strains containing excision-defective mutations in any of nine genes in combination with the cdc9 mutation, the absence of low-molecular-weight DNA at the nonpermissive temperature after ultraviolet treatment suggests that these mutants are incision defective, whereas the presence of low-molecular-weight DNA indicates that the mutants are defective in a step after incision. With rad1, rad2, rad3, rad4, and rad10 mutants, the molecular weight of the DNA remained unchanged after ultraviolet irradiation and incubation at the restrictive temperature, despite the presence of the cdc9 mutation; these mutants are therefore incision defective. Low-molecular-weight DNA was observed in rad14 cdc9 and rad16 cdc9 strains. With the rad16 strain, the accumulation of low-molecular-weight DNA correlated with the amount of excision taking place, whereas in the rad14 mutant strain, no evidence of dimer removal was obtained. Therefore, rad14 is likely to be defective in a step after incision.     

Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle

Thymidine kinase and dihydrofolate reductase mRNA levels and enzyme activities were determined in two temperature-sensitive cell lines, tsAF8 and ts13, that growth arrest in the G1 phase of the cell cycle at the restrictive temperature. The levels of thymidine kinase mRNA and enzyme activity increased markedly in both cell lines serum stimulated from quiescence at the permissive temperature. At the nonpermissive temperature, the levels of thymidine kinase mRNA and enzyme activity remain at the low levels of quiescent G0 cells. The levels of dihydrofolate reductase mRNA as well as the enzyme activity also increase when both cell lines are serum stimulated at the permissive temperature. When ts13 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity declines rapidly and dihydrofolate reductase mRNA is below detectable levels. On the contrary, when tsAF8 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity increases and mRNA levels are detectable slightly above G0 levels, even though the cells are blocked in the G1 phase. Studies with 2 other cDNA clones (one with an insert whose expression is cell cycle dependent and the other with an insert whose expression is not cell cycle dependent) indicate that the results are not due to aspecific toxicity or the effect of temperature. We conclude that the expression of different genes is affected differently by the ts block in G1, even when these genes are all growth-related.     

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB^A116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB^A116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB^A116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.     

A locus affecting nucleoid segregation in Salmonella typhimurium.

Thirteen temperature-sensitive lethal mutations of Salmonella typhimurium map near metC at 65 min and form the clmF (conditional lethal mutation) locus. The mutations in this region were ordered by three-point transduction crosses. After a shift to the nonpermissive temperature, many of these clmF mutants failed to complete the segregation of nucleoids into daughter cells; daughter nucleoids appeared incompletely separated and asymmetrically positioned within cells. Some clmF mutants showed instability of F' episomes at permissive growth temperatures yet showed no detectable defect with smaller multicopy plasmids such as pSC101 or pBR322. In addition, many of the clmF mutants rapidly lost viability yet continued DNA replication at the nonpermissive temperature. These results suggest that the clmF locus encodes at least one indispensable gene product that is required for faithful partitioning of the bacterial nucleoid and F-plasmid replicons.     

Specific inhibition of phospholipid synthesis in plsA mutants of Escherichia coli.

plsA mutants of Escherichia coli are temperature-sensitive strains which possess two enzymes of abnormal thermolability, sn-glycerol 3-phosphate acyltransferase and adenylate kinase. Phospholipid synthesis is inhibited after shift of plsA mutants to temperatures at the lower end of the nonpermissive temperature range. This inhibition is not due to inactivation of the adenylate kinase activity since nucleic acid (and hence adenosine 5'-triphosphate) synthesis is inhibited only slightly. These results show that in vivo inactivation of the sn-glycerol 3-phosphate acyltransferase can be observed under conditions which allow normal adenylate kinase function.     

Replication of the Bacteriocinogenic Plasmid Clo DF13 in Thermosensitive Escherichia coli Mutants Defective in Initiation or Elongation of Deoxyribonucleic Acid Replication

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.     

Roles of Replication Protein-a Subunits 2 and 3 in DNA Replication Fork Movement in Saccharomyces Cerevisiae

Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (RPA2 and 3) that may cooperate with RPA1 to bind ssDNA in a higher-order mode. To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an assortment of heat-lethal mutations in the genes encoding RPA2 and RPA3. At the permissive temperature, the mutants show a range of effects on DNA replication fidelity and sensitivities to UV and MMS. At the nonpermissive temperature, four out of five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block. In contrast, the fifth RPA2 mutant and all RPA3 mutants are able to complete at least one round of DNA replication at the nonpermissive temperature. The effect of these mutations on the stability of the RPA complex was tested using a coprecipitation assay. At the nonpermissive temperature, we find that RPA1 and RPA2 are dissociated in the fast-stop mutants, but not in the slow-stop mutants. Thus, replication fork movement in vivo requires the association of at least two subunits of RPA. This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode.