SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells.

We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site. Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum. We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions. In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions. Replication efficiency was 29% in these conditions. SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum.     

Expression of a Saccharomyces cerevisiae photolyase gene in Escherichia coli.

A 3.3-kilobase PvuII fragment carrying the PHR1 gene of Saccharomyces cerevisiae has been cloned into an Escherichia coli expression vector and introduced into E. coli strains deficient in DNA photolyase. Complementation of the E. coli phr-1 mutation was observed, strongly suggesting that the yeast PHR1 gene encodes a DNA photolyase.     

Effects of accessory proteins on the bypass of a cis-syn thymine-thymine dimer by Saccharomyces cerevisiae DNA polymerase eta

Among several hypotheses to explain how translesion synthesis (TLS) by DNA polymerase eta (pol eta) suppresses ultraviolet light-induced mutagenesis in vivo despite the fact that pol eta copies DNA with low fidelity, here we test whether replication accessory proteins enhance the fidelity of TLS by pol eta. We first show that the single-stranded DNA binding protein RPA, the sliding clamp PCNA, and the clamp loader RFC slightly increase the processivity of yeast pol eta and its ability to recycle to new template primers. However, these increases are small, and they are similar when copying an undamaged template and a template containing a cis-syn TT dimer. Consequently, the accessory proteins do not strongly stimulate the already robust TT dimer bypass efficiency of pol eta. We then perform a comprehensive analysis of yeast pol eta fidelity. We show that it is much less accurate than other yeast DNA polymerases and that the accessory proteins have little effect on fidelity when copying undamaged templates or when bypassing a TT dimer. Thus, although accessory proteins clearly participate in pol eta functions in vivo, they do not appear to help suppress UV mutagenesis by improving pol eta bypass fidelity per se.     

Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli

The Saccharomyces cerevisiae glycerol pathway (GPD1 and GPP2) was evolved in vivo in Escherichia coli. The central metabolism of E. coli was engineered to link glucose consumption and glycerol production. The engineered strain was evolved in a chemostat culture and a high glycerol producer was rapidly obtained. The evolution of the strain was associated to a deletion between GPD1 and GPP2, resulting in the production of a fusion protein with both glycerol-3-P dehydrogenase and glycerol-3-P phosphatase activities. The higher efficiency of the fusion protein was due to partial glycerol-3-P channeling between the two active sites. The evolved strain produces glycerol from glucose at high yield, concentration and productivity.     

Saccharomyces cerevisiae acquires resistance to 2-deoxyglucose at a very high frequency.

We have found that Saccharomyces cerevisiae acquires spontaneously increasing resistance to 2-deoxyglucose at a very high frequency. This finding allows the easy isolation of different types of resistant strains of interest for metabolic studies with 2-deoxyhexoses. On the other hand, it sounds a note of caution in the widespread use of 2-deoxyglucose as a selective agent for the isolation of yeast mutants with impaired hexose transport or phosphorylation systems.     

Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer

The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis–syn cyclobutane thymine–uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3′–5′)-2′-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis–syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase η was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol η bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C→T transition because the original sequence is TC.     

Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae.

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.     

Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells

In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNA(Tyr) with tyrosine. Resulting d-Tyr-tRNA(Tyr) can be hydrolyzed by a d-Tyr-tRNA(Tyr) deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be exacerbated by the inactivation of the gene encoding d-Tyr-tRNA(Tyr) deacylase. In addition to the already documented case of d-tyrosine, positive responses were obtained with d-tryptophan, d-aspartate, d-serine, and d-glutamine. In agreement with this observation, production of d-Asp-tRNA(Asp) and d-Trp-tRNA(Trp) by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was established in vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNA(Tyr) deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation of d-aminoacyl-tRNA molecules and that d-Tyr-tRNA(Tyr) deacylase has a specificity broad enough to recycle any of these molecules. The same strategy of screening was applied using Saccharomyces cerevisiae, the tyrosyl-tRNA synthetase of which also produces d-Tyr-tRNA(Tyr), and which, like E. coli, possesses a d-Tyr-tRNA(Tyr) deacylase activity. In this case, inhibition of growth by the various 19 d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects of d-tyrosine and d-leucine were reinforced upon deprivation of the deacylase. This observation suggests that, in yeast, at least two d-amino acids succeed in being transferred onto tRNAs and that, like in E. coli, the resulting two d-aminoacyl-tRNAs are substrates of a same d-aminoacyl-tRNA deacylase.     

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.     

The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.     

Suppression of Escherichia coli alkB mutants by Saccharomyces cerevisiae genes.

The alkB gene is one of a group of alkylation-inducible genes in Escherichia coli, and its product protects cells from SN2-type alkylating agents such as methyl methanesulfonate (MMS). However, the precise biochemical function of the AlkB protein remains unknown. Here, we describe the cloning, sequencing, and characterization of three Saccharomyces cerevisiae genes (YFW1, YFW12, and YFW16) that functionally complement E. coli alkB mutant cells. DNA sequence analysis showed that none of the three gene products have any amino acid sequence homology with the AlkB protein. The YFW1 and YFW12 proteins are highly serine and threonine rich, and YFW1 contains a stretch of 28 hydrophobic residues, indicating that it may be a membrane protein. The YFW16 gene turned out to be allelic with the S. cerevisiae STE11 gene. STE11 is a protein kinase known to be involved in pheromone signal transduction in S. cerevisiae; however, the kinase activity is not required for MMS resistance because mutant STE11 proteins lacking kinase activity could still complement E. coli alkB mutants. Despite the fact that YFW1, YFW12, and YFW16/STE11 each confer substantial MMS resistance upon E. coli alkB cells, S. cerevisiae null mutants for each gene were not MMS sensitive. Whether these three genes provide alkylation resistance in E. coli via an alkB-like mechanism remains to be determined, but protection appears to be specific for AlkB-deficient E. coli because none of the genes protect other alkylation-sensitive E. coli strains from killing by MMS.     

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.     

Genetic activity of carbamate herbicides in Escherichia coli and Saccharomyces cerevisiae

DNA-damaging activity and herbicide-induction of gene point mutations, and intragenic mitotic recombination were studied in bacteria and the yeast tester strains. Herbicide (eptam, triallate, tillam, surpass) were not effective in DNA-damaging and mitotic recombination tests. Of the 4 chemicals, only eptam was strongly mutagenic. Dose response curves for eptam differed in bacteria and yeast; maximum mutagenic activity was registered in bacteria at 5 mg/l. Maximum yield of prototrophs was observed after 2 h incubation time. Triallate was moderate, tillam and surpass being weak mutagens for the strains used.     

Mutational inactivation of the Saccharomyces cerevisiae RAD4 gene in Escherichia coli.

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. When plasmids containing the wild-type gene were transformed into various Escherichia coli strains, transformation frequencies were drastically reduced. Most plasmids recovered from transformants showed deletions or rearrangements. A minority of plasmids recovered from E. coli HB101 showed no evidence of deletion or rearrangement, but when they were transformed into S. cerevisiae on centromeric vectors, little or no complementation of the UV sensitivity of rad4 mutants was observed. Deliberate insertional mutagenesis of the wild-type RAD4 allele before transformation of E. coli restored transformation to normal levels. Plasmids recovered from these transformants contained an inactive rad4 allele; however, removal of the inserted DNA fragment restored normal RAD4 function. These experiments suggest that expression of the RAD4 gene is lethal to E. coli and show that lethality can be prevented by inactivation of the gene before transformation. Stationary-phase cultures of some strains of E. coli transformed with plasmids containing an inactivated RAD4 gene showed a pronounced delay in the resumption of exponential growth, suggesting that the mutant (and, by inference, possibly wild-type) Rad4 protein interferes with normal growth control in E. coli. The rad4-2, rad4-3, and rad4-4 chromosomal alleles were leaky relative to a rad4 disruption mutant. In addition, overexpression of plasmid-borne mutant rad4 alleles resulted in partial complementation of rad4 strains. These observations suggest that the Rad4 protein is relatively insensitive to mutational inactivation.     

Expression of the Bacillus subtilis levanase gene in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for the inulin hydrolyzing enzyme levanase which was previously cloned from Bacillus subtilis was fused to the tac-promoter. Overexpression in Escherichia coli resulted in high amounts of intracellularly produced levanase (up to 20 U mg-1). After removal of the bacterial 5' sequences, the levanase gene was also cloned into a yeast expression vector based on the PGK-promoter. Clones containing the intact levanase gene including the bacterial signal sequence gave rise to synthesis of active levanase by Saccharomyces cerevisiae transformants. A considerable amount of levanase protein was found in the culture medium (around 0.5 U ml-1) indicating efficient secretion of B. subtilis levanase from yeast.     

Metalloenzymes in DNA repair. Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1.

Escherichia coli endonuclease IV and its Saccharomyces cerevisiae homologue Apn1, two DNA repair enzymes for free radical damages, were previously shown to be inactivated by metal-chelating agents. In the present study, atomic absorption spectrometry of endonuclease IV revealed the presence of 2.4 zinc and 0.7 manganese atoms, whereas Apn1 contained 3.3 zinc atoms and no significant manganese. EDTA-inactivated endonuclease IV retained 0.7 zinc atom but little detectable manganese. ZnCl2 reactivated 1,10-phenanthroline-treated Apn1, but was ineffective with endonuclease IV treated with either 1,10-phenanthroline or EDTA. In contrast, enzymatic activity was restored to both enzymes after EDTA treatment by incubation with CoCl2 and to a lesser extent by MnCl2. Endonuclease IV, reactivated with CoCl2 or MnCl2, regained all of the activities characteristic of the native enzyme. MnCl2 was as effective as CoCl2 at restoring activity to the 1,10-phenanthroline-treated enzymes. The results indicate that intrinsic metals play critical roles in both endonuclease IV and Apn1 and that manganese may perform a special function in endonuclease IV. Possible mechanistic roles for the metals in these DNA repair enzymes are discussed.     

Expression of the Saccharomyces cerevisiae PIS gene and synthesis of phosphatidylinositol in Escherichia coli.

Expression of the Saccharomyces cerevisiae PIS gene encoding phosphatidylinositol synthase in Escherichia coli was achieved by inserting its coding sequence into lacZ on pUC8. The fused gene encoded a phosphatidylinositol synthase whose amino-terminal three amino acids had been replaced by the amino-terminal five amino acids of E. coli beta-galactosidase. E. coli cells bearing this recombinant plasmid produced a significant level of phosphatidylinositol synthase in the presence of a lacZ inducer, isopropylthio-beta-D-galactopyranoside. When the culture medium was supplemented with myo-inositol and isopropylthio-beta-D-galactopyranoside, the cells accumulated a substantial amount of phosphatidylinositol in their membranes. When a saturating level of myo-inositol was added, phosphatidylinositol constituted about 4% of the total phospholipids. Phosphatidylinositol accumulation occurred at the expense of phosphatidylglycerol. The ratio of phosphatidylethanolamine to total acidic phospholipids remained constant. The growth rate of phosphatidylinositol-containing E. coli cells did not differ significantly from that of cells with the normal phospholipid composition.     

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.