SOS Conditions Research Articles

Effects of Oak Leaf Extract, Biofertilizer, and Soil Containing Oak Leaf Powder on Tomato Growth and Biochemical Characteristics under Water Stress Conditions

Drought stress is one of the most significant abiotic stresses on the sustainability of global agriculture. The finding of natural resources is essential for decreasing the need for artificial fertilizers and boosting plant growth and yield under water stress conditions. This study used a factorial experimental design to investigate the effects of oak leaf extract, biofertilizer, and soil containing oak leaf powder on the growth and biochemical parameters of four tomato genotypes under water stress throughout the pre-flowering and pre-fruiting stages of plant development. The experiment had two components. The first component represented the genotypes (two sensitive and two tolerant), while the second component represented the treatment group, which included irrigated plants (SW), untreated and stressed plants (SS), treated plants with oak leaf powder and stressed (SOS), treated plants with oak leaf powder and oak leaf extract and stressed (SOES), and treated plants with oak leaf powder and biofertilizers and stressed (SOBS). When compared with irrigated or control plants, drought stress under the treatments of SS, SOS, SOES, and SOBS conditions at two stages and their combination significantly lowered shoot length (12.95%), total fruit weight per plant (33.97%), relative water content (14.05%), and total chlorophyll content (26.30%). The reduction values for shoot length (17.58%), shoot fresh weight (22.08%), and total fruit weight per plant (42.61%) were significantly larger in two sensitive genotypes compared with tolerant genotypes, which recorded decreasing percentages of 8.36, 8.88, and 25.32% for shoot length, shoot fresh weight, and total fruit weight per plant, respectively. Root fresh weight and root dry weight of genotypes treated with SS, SOS, SOES, and SOBS, on the other hand, increased in comparison with control plants. Tomato fruits from stressed plants treated with SS, SOS, SOES, and SOBS had considerably higher levels of titratable acidity, ascorbic acid, and total phenolic compounds than irrigated plants during all stress stages. Under water stress conditions, the addition of oak leaf powder to soil, oak leaf extract, and biofertilizer improved the biochemical content of leaves in all genotypes. Furthermore, leaf lipid peroxidation was lower in plants treated with SOES and SOBS, and lower in the two tolerant genotypes than in the two susceptible genotypes. In conclusion, the application of SOS, SOES, and SOBS demonstrated a slight decrease in some morpho-physiological and fruit physicochemical traits compared with SS treatment. However, the application of oak leaf powder and oak leaf extract can be described as novel agricultural practices because they are low-cost, easy to use, time-consuming, and can meet the growing demands of the agricultural sector by providing environmentally sustainable techniques for enhancing plant resistance to abiotic stress. The usage of the combination of leaf crude extract, oak leaf powder, and arbuscular mycorrhizal fungus should be investigated further under stress conditions.

A dual-function phage regulator controls the response of cohabiting phage elements via regulation of the bacterial SOS response.

SummaryListeria monocytogenes strain 10403S harbors two phage elements in its chromosome; one produces infective virions and the other tailocins. It was previously demonstrated that induction of the two elements is coordinated, as they are regulated by the same anti-repressor. In this study, we identified AriS as another phage regulator that controls the two elements, bearing the capacity to inhibit their lytic induction under SOS conditions. AriS is a two-domain protein that possesses two distinct activities, one regulating the genes of its encoding phage and the other downregulating the bacterial SOS response. While the first activity associates with the AriS N-terminal AntA/AntB domain, the second associates with its C-terminal ANT/KilAC domain. The ANT/KilAC domain is conserved in many AriS-like proteins of listerial and non-listerial prophages, suggesting that temperate phages acquired such dual-function regulators to align their response with the other phage elements that cohabit the genome.

Remediation of triazole, anilinopyrimidine, strobilurin and neonicotinoid pesticides in polluted soil using ozonation and solarization

This study aimed to investigate the effectiveness of ozonation and solarization techniques for the removal of different types of pesticides from soil during the summer season. The effect of two experimental parameters (temperature and ozone application mode) on the pesticide degradation was evaluated. The results showed that solarization (S), solarization with surface ozonation (SOS), and solarization with deep ozonation (SOD) enhanced pesticide degradation rates in comparison with the control (untreated soil, C). The triazole, anilinopyrimidine, strobilurin and neonicotinoid pesticides showed similar behaviour under S and SOS conditions. The highest decrease was found in SOD, indicating the significant effect of temperature and ozone application mode on the efficiency of the ozonation treatment. Thus, a higher soil temperature and a longer accumulated time at high temperature in treatments S, SOS and SOD were observed due to solarization process. In addition, the removal efficiency was enhanced with exposure time. Finally, the main 15 transformation products were identified during SOD treatment. The results suggest that solarization combined with ozonation techniques allows decontamination of soil containing pesticide residues.

Robust model predictive control for hypersonic vehicle with state‐dependent input constraints and parameter uncertainty

AbstractIn this article, a robust model predictive control strategy based on sum‐of‐squares (SOS‐RMPC) is proposed for hypersonic vehicle with state‐dependent input constraints and uncertain parameters. Based on feedback linearization model of hypersonic vehicle, the polytopic linear parameter varying error model with bounded disturbance is established for reference trajectory tracking. The real limits of the actual control inputs are transformed into the constraints of the virtual inputs equivalently with the state‐dependent nonlinear functions. Further, the multivariate linear fitting is used to approximate the state‐dependent input constraints into polynomials. A weighted composite virtual control is formulated as the combination of unconstrained control and auxiliary control. SOS technique is introduced to cast the polynomial constraints into the convex matrix SOS conditions via linear matrix inequality. The virtual control law can be obtained by solving a SOS‐RMPC convex optimization with infinite horizon. The invariant set is designed for the undisturbed error states and the norm‐bounding theory is applied to ensure that the predictive error states with disturbance can remain in the same invariant set. The real control law is obtained by inverse control. The simulation verifies the performance of the designed controller.

Post‐transcriptional deregulation of the tisB/istR‐1 toxin–antitoxin system promotes SOS‐independent persister formation in Escherichia coli

Bacterial dormancy is a valuable strategy to endure unfavourable conditions. The term 'persister' has been coined for cells that tolerate antibiotic treatments due to reduced cellular activity. The type I toxin-antitoxin system tisB/istR-1 is linked to persistence in Escherichia coli, because toxin TisB depolarizes the inner membrane and causes ATP depletion. Transcription of tisB is induced upon activation of the SOS response by DNA-damaging drugs. However, translation is repressed both by a 5' structure within the tisB mRNA and by RNA antitoxin IstR-1. This tight regulation limits TisB production to SOS conditions. Deletion of both regulatory RNA elements produced a 'high persistence' mutant, which was previously assumed to depend on stochastic SOS induction and concomitant TisB production. Here, we demonstrate that the mutant generates a subpopulation of growth-retarded cells during late stationary phase, likely due to SOS-independent TisB accumulation. Cell sorting experiments revealed that the stationary phase-derived subpopulation contains most of the persister cells. Collectively our data show that deletion of the regulatory RNA elements uncouples the persister formation process from the intended stress situation and enables the formation of TisB-dependent persisters in an SOS-independent manner.

The hipBAXn operon from Xenorhabdus nematophila functions as a bonafidetoxin-antitoxin module.

Here, for the first time, we have investigated the hipBAXn toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipAXn toxin and HipBXn antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipAXn toxin, hipBXn antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipAXn toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipAXn toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157bp upstream region of the hipBAXn TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipAXn toxin, HipBXn antitoxin protein, and 157bp promoter region. Results showed that the hipBAXn TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions. KEY POINTS: •Functional characterization of hipBA Xn TA module from Xenorhabdus nematophila. •hipBA Xn TA module is a functional type II TA module. •Transcriptional characterization of hipBA Xn TA module. •hipBA Xn TA module is a well regulated TA module. Graphical abstract.

Xenocin Export by the Flagellar Type III Pathway in Xenorhabdus nematophila

The xenocin operon of Xenorhabdus nematophila consists of xciA and ximB genes encoding a 64-kDa xenocin and 42-kDa immunity protein to kill competing microbes in the insect larva. The catalytic domain of xenocin has RNase activity and is responsible for its cytotoxicity. Under SOS conditions, xenocin is produced with immunity protein as a complex. Here, we show that xenocin and immunity protein complex are exported through the flagellar type III system of X. nematophila. Secretion of xenocin complex was abolished in an flhA strain but not in an fliC strain. The xenocin operon is not linked to the flagellar operon transcriptionally. The immunity protein is produced alone from a second, constitutive promoter and is targeted to the periplasm in a flagellum-independent manner. For stable expression of xenocin, coexpression of immunity protein was necessary. To examine the role of immunity protein in xenocin export, an enzymatically inactive protein was produced by site-directed mutagenesis in the active site of the catalytic domain. Toxicity was abolished in D535A and H538A variants of xenocin, which were expressed alone without an immunity domain and secreted in the culture supernatant through flagellar export. Secretion of xenocin through the flagellar pathway has important implications in the evolutionary success of X. nematophila.

Structural and Functional Interferences from a Molecular Structural Model of Xenocin<i> </i>Toxin from<i> Xenorhabdus nematophila</i>

Bacteriocins constitute the most abundant and diverse family of microbial defense systems. They are pro- duced and secreted to inhib it the gro wth of closely related, co mpeting bacterial species inhabiting a co mmon ecological niche. One such bacteriocin designated as xenocin, is produced by entomopathogenic bacteriu m Xenorhabdus nematophila. In our earlier study, we have shown its regulation under SOS conditions and its activity against wide range of bacteria iso- lated fro m the gut of insect. In this study three dimensional structure of xenocin has been deciphered with the assistance of automated homology modelling and verified by VERIFY-3D program as well as Ramachandran plot 2.0. Three domain organisation; Translocation (T), Receptor (R) and Catalytic do main (C) has been o-0.058 served and their structures were studied by CHIM ERA software (UCSF). Protein disorder structure and Average Area Buried Upon folding (AABUF) in first 100 amino acid residues of Translocation domain is determined by Globplot and ProtScale software respectively. Conserved amino acid residues in the putative active site of the catalytic domain of xenocin have been deciphered with mult iple se- quence alignment.

A mixed integer approach for time-dependent gas network optimization

A gas network consists of pipes to transport the gas from the suppliers to the consumers. Due to friction with the pipe walls, gas pressure gets lost. Compressors compensate this pressure loss at the cost of consuming fuel gas. The aim of gas network optimization is to minimize the fuel gas consumption of the compressors in such a way that the contracts with consumers and suppliers are fulfilled. This problem leads to a very complex mixed integer nonlinear optimization problem. We present a linear approach which concentrates on time-dependent and discrete aspects. The nonlinearities are approximated by piece-wise linear functions using the concept of SOS constraints. We develop a branch-and-cut algorithm which has the potential to guarantee global optimality of the linearized problem where the nonlinearities are approximated within a given accuracy. We include an adequate handling of the SOS conditions and a separation algorithm for switching processes of compressors. A simulated annealing algorithm is included yielding feasible solutions. Our computational results show the success of our approach in this challenging field of gas network optimization.

Transcriptional Analysis and Functional Characterization of a Gene Pair Encoding Iron-Regulated Xenocin and Immunity Proteins of Xenorhabdus nematophila

We describe a two-gene cluster encoding a bacteriocin, xenocin, and the cognate immunity protein in the insect-pathogenic bacterium Xenorhabdus nematophila, which infects and kills larval stages of the common crop pest Helicoverpa armigera. The two genes, xcinA and ximB, are present in the genome as a single transcriptional unit, which is regulated under SOS conditions. The stress-inducible promoter was activated by mitomycin C, glucose, and Fe(3+) depletion and at an elevated temperature when it was tested in Escherichia coli cells. Expression of the xenocin protein alone in E. coli inhibited the growth of this organism. The growth inhibition was abolished when the immunity protein was also present. A recombinant xenocin-immunity protein complex inhibited the growth of E. coli indicator cells when it was added exogenously to a growing culture. Xenocin is an endoribonuclease with an enzymatically active C-terminal domain. Six resident bacterial species (i.e., Bacillus, Enterobacter, Enterococcus, Citrobacter, Serratia, and Stenotrophomonas species) from the H. armigera gut exhibited sensitivity to recombinant xenocin when the organisms were grown under iron-depleted conditions and at a high temperature. Xenocin also inhibited the growth of two Xenorhabdus isolates. This study demonstrates that Fe(3+) depletion acts as a common cue for synthesis of xenocin by X. nematophila and sensitization of the target strains to the bacteriocin.

Evidence for Escherichia coli polymerase II mutagenic bypass of intrastrand DNA crosslinks

The mutagenic potentials of DNAs containing site- and stereospecific intrastrand DNA crosslinks were evaluated in Escherichia coli cells that contained a full complement of DNA polymerases or were deficient in either polymerases II, IV, or V. Crosslinks were made between adjacent N 6 - N 6 adenines and consisted of R, R- and S, S-butadiene crosslinks and unfunctionalized 2-, 3-, and 4-carbon tethers. Although replication of single-stranded DNAs containing the unfunctionalized 3- and 4-carbon tethers were non-mutagenic in all strains tested, replication past all the other intrastrand crosslinks was mutagenic in all E. coli strains, except the one deficient in polymerase II in which no mutations were ever detected. However, when mutagenesis was analyzed in cells induced for SOS, mutations were not detected, suggesting a possible change in the overall fidelity of polymerase II under SOS conditions. These data suggest that DNA polymerase II is responsible for the in vivo mutagenic bypass of these lesions in wild-type E. coli.

Effects of the C4‘-Oxidized Abasic Site on Replication in Escherichia coli. An Unusually Large Deletion Is Induced by a Small Lesion

The C4'-oxidized abasic site (C4-AP) is produced in DNA as a result of oxidative stress by a variety of agents. For instance, the lesion accounts for approximately 40% of the DNA damage produced by the antitumor antibiotic bleomycin. The effect of C4-AP on DNA replication in Escherichia coli was determined using the restriction endonuclease and postlabeling (REAP) method. Three-nucleotide deletion products are the sole products observed following replication of plasmids containing C4-AP under SOS conditions in wild-type cells. Full-length products are formed in varying amounts depending upon the local sequence in wild-type cells under non-SOS-induced conditions. The "A-rule" is followed for the formation of substitution products. C4-AP is the first example of a DNA lesion that produces significant levels of three-nucleotide deletions in a variety of sequence contexts. Experiments carried out in cells lacking specific polymerases reveal that formation of three-nucleotide deletion products results from a coordinated effort involving pol II and pol IV. This is the first example in which these SOS inducible polymerases are shown to work in concert during lesion bypass. Three-nucleotide deletions are not observed during the replication of other abasic lesions, and are rarely produced by bulky adducts. The effect of C4-AP on DNA replication suggests a significant role for this lesion in the cytotoxicity of bleomycin. Formation of the C4-AP lesion may also be responsible for the formation of mutant proteins containing single-amino acid deletions that exhibit altered phenotypes.

Error-free Recombinational Repair Predominates over Mutagenic Translesion Replication in E. coli

Tolerance mechanisms are important in the ability of cells to cope with DNA damage. In E. coli, the two main damage tolerance mechanisms are recombinational repair (RR) and translesion replication (TLR). Here we show that RR effectively repairs gaps opposite DNA lesions. When both mechanisms are functional, RR predominates over TLR, being responsible for 86% of the repair events. This predominance of RR is determined by the high concentration of RecA present under SOS conditions, which causes a differential inhibition of TLR. Further inhibition of TLR is caused by the RecA-catalyzed strand exchange reaction of RR. This molecular hierarchy in the tolerance of DNA lesions ensures that the nonmutagenic RR predominates over the mutagenic TLR, thereby contributing to genetic stability.

SOS-dependent A→G transitions induced by hydroxyl radical generating system hypoxanthine/xanthine oxidase/Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mp18 phage DNA

Gas chromatography/isotope dilution–mass spectrometry with selected ion monitoring (GC/IDMS–SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C→A (∼20-fold increase in SOS-conditions), G→A (9-fold increase) and G→C (4.5-fold increase). Very few G→T transitions were found. A particularly large group of A→G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A→G transitions in SOS-induced bacteria.

Mutation spectra of TA*, the major photoproduct of thymidylyl-(3'5')-deoxyadenosine, in Escherichia coli under SOS conditions.

The biological activity of TA*, the major photoproduct of thymidylyl-(3',5')-deoxyadenosine, has remained speculative since it was identified a decade ago. To determine the mutagenicity of TA* in Escherichia coli, we constructed the replicative form of an M13mp18-derived phage containing TA* in the (-)-strand by polymerase-catalyzed elongation of a TA*-containing 49mer opposite a uracil-containing (+)-strand of the phage. The in vitro synthesis mixture was transfected into an ung+, phr- E.coli host and the progeny were screened with a hybridization probe unique for the (-)-strand. TA* was found to block DNA replication substantially in the absence of SOS, but under SOS, TA* was bypassed more efficiently and was highly mutagenic. Among 56 analyzed (-)-strand progeny from two transfections, 46 (82%) were mutants, including six (11%) tandem mutants. The most abundant mutation was a 3'A-->T substitution (31/46, 56%). The possible biological consequences of TA* formation in the highly conserved TATA box consensus sequence on gene expression are discussed in light of the mutagenicity of TA*.

Contrasting structural impacts induced by cis-syn cyclobutane dimer and (6-4) adduct in DNA duplex decamers: implication in mutagenesis and repair activity.

The relative biological importance of cis--syn cyclobutane dimer and pyrimidine(6-4)pyrimidone photoadduct ([6-4] photoadduct) appears to be dependent on the biological species, dipyrimidine sites and local conformational variation induced at the damaged sites. The single-strained deoxynucleotide 10-mers containing the site-specific (6-4) adduct or cis--syn cyclobutane dimer of thymidylyl(3'-->5')-thymidine were generated by direct photolysis of d(CGCATTACGC) with UVC (220-260 nm) irradiation or UVB (260-320 nm) photosensitization. Three-dimensional structures of the duplex cis--syn and (6-4) decamers of d(CGCATTACGC)xd(GCGTAATGCG) were determined by NMR spectroscopy and the relaxation matrix refinement method. The NMR data and structural calculations establish that Watson-Crick base pairing is still intact at the cis--syn dimer site while the hydrogen bonding is absent at the 3'-side of the (6-4) lesion where the T-->C transition mutation is predominantly targeted. Overall conformation of the duplex cis--syn decamer was B-DNA and produced a 9 degree bending in the DNA helix, but a distinctive base orientation of the (6-4) lesion provided a structural basis leading to 44 degree helical bending. The observed local structure and conformational rigidity at the (6-4) adduct of the thymidylyl(3'-5')-thymidine (T-T [6-4]) lesion site suggest the potential absence of hydrogen bonding at the 3' sides of the (6-4) lesion with a substituted nucleotide during replication under SOS conditions. Contrasting structural distortions induced ny the T-T (6-4) adduct with respect to the T-T cis--syn cyclobutane pyrimidine photodimer may explain the large differences in mutation spectrum and repair activities between them.

DNA polymerase II of Escherichia coli in the bypass of abasic sites in vivo.

The function of DNA polymerase II of Escherichia coli is an old question. Any phenotypic character that Pol II may confer upon the cell has escaped detection since the polymerase was discovered 24 yr ago. Although it has been shown that Pol II enables DNA synthesis to proceed past abasic sites in vitro, no role is known for it in the bypass of those lesions in vivo. From a study of phage S13 single-stranded DNA, we now report SOS conditions under which Pol II is needed for DNA synthesis to proceed past abasic sites with 100% efficiency in vivo. Overproduction of the GroES+L+ heat shock proteins, which are members of a ubiquitous family of molecular chaperones, eliminated this requirement for Pol II, which may explain why the role of Pol II in SOS repair had eluded discovery. Mutagenesis accompanied SOS bypass of abasic sites when the original occupant had been cytosine but not when it had been thymine; the quantitative difference is shown to imply that adenine was inserted opposite the abasic sites at least 99.7% of the time, which is an especially strict application of the A-rule. Most, but not all, spontaneous mutations from Rifs to Rifr, whether in a recA+ or a recA(Prtc) cell, require Pol II; while this suggests that cryptic abasic lesions are a likely source of spontaneous mutations, it also shows that such lesions cannot be the exclusive source.

Accuracy of Replication Past the T-C (6-4) Adduct

The thymine cytosine pyrimidine-pyrimidone (6-4) adduct has variously been predicted to be among the most and among the least mutagenic of the ultraviolet light photoproducts. We have therefore investigated the frequency and accuracy of DNA replication past this lesion, using a single-stranded M13mp7-based vector with a uniquely located example of this lesion transfected into SOS induced and uninduced cells of a uvr A6 strain of Escherichia coli. Both the UVC T-C (6-4) adduct and its Dewar valence (UVB) photoisomer were studied. Random samples from non-selective collections of progeny phage were sequenced to determine the nature of the replication events that occurred at or near the site of template damage under SOS conditions. The UVC (6-4) adduct was found to be much less mutagenic than its T-T counterpart, but still much more mutagenic than a cyclobutane dimer; 34% (71 out of 206) of all bypass events yielded mutations, of which all were targeted and 80% (57 out of 71) were 3′ C → T transitions. The Dewar valence photoisomer exhibited reduced specificity and enhanced mutagenicity; 79% (183 out of 233) of the phage progeny were mutants, of which all but one were targeted and 45% (83 out of 183) were 3′ C → T transitions. For the most part, these results are consistent with a model postulating base-pairing between the pyrimidinone (of either the C or T variety) and guanine, via hydrogen bonds at N-3 and O-2 in the UVC, but not the Dewar, isomer. The occurrence of the 3′ C → T transitions, not predicted by this model, shows however that the absence of a methyl group at C-5 also has a significant influence on mutation induction. Both isomers were efficient blocks to replication; less than 1% of these vectors could be replicated in uninduced cells. Following SOS induction the frequency of bypass increased to 24·5% and 12·5 for the UVC and the Dewar isomers, respectively.

In vivo evidence that UV-induced C .fwdarw. T mutations at dipyrimidine sites could result from the replicative bypass of cis-syn cyclobutane dimers or their deamination products

The major mutations induced by UV light are C-->T transitions at dipyrimidines and arise from the incorporation of A opposite the C of dipyrimidine photoproducts. The incorporation of A has most often been explained by the known preference of a polymerase to do so opposite noninstructional DNA damage such as an abasic site (A rule). There are also mechanisms that suppose, however, that cis-syn dipyrimidine photodimers are instructional. In one such mechanism (tautomer bypass), the incorporation of A is directed by the tautomer of a C of a dimer that is equivalent in base-pairing properties to U [Person et al. (1974) Genetics 78, 1035-1049]. In another mechanism (deamination bypass), the incorporation of A is directed by a U of a dimer that results from the deamination of the C of a dimer [Taylor & O'Day (1990) Biochemistry 29, 1624-1632]. The viability of these mechanisms was tested by obtaining the mutation spectrum of a TU dimer in Escherichia coli by application of a standard method for site-directed mutagenesis. To this end, a 41-mer containing a site-specific TU dimer was constructed via ligation of a dimer-containing decamer that was produced by triplet-sensitized irradiation and used to prime DNA synthesis on a uracil-containing (+) strand of an M13 clone containing a double mismatch opposite the dimer. The reaction mixture was used to transfect a uracil glycosylase proficient, photoproduct repair deficient E. coli host, and all progeny phage weakly hybridizing to the parental (+) or (-) strands were sequenced. Under non-SOS conditions the TU dimer almost completely blocked replication, while under SOS conditions it directed the incorporation of two As with much higher specificity (96%) than would an abasic site. The implications of these results to the mechanism of the UV-induced TC-->TT mutation, and by extension to the CT-->TT, CC-->TC, CC-->CT, and the tandem CC-->TT mutations, are discussed.

Further evidence that transposition of Tn5 in Escherichia coli is strongly enhanced by constitutively activated RecA proteins.

We have shown that excision and transposition of Tn5 in Escherichia coli are greatly increased by recA(Prtc) genes, which encode constitutively activated RecA proteins (C.-T. Kuan, S.-K. Liu, and I. Tessman, Genetics 128:45-57, 1991). Contrary results, showing a significant decrease in Tn5 transposition under SOS conditions, were subsequently reported (M. D. Weinreich, J. C. Makris, and W. S. Reznikoff, J. Bacteriol. 173:6910-6918, 1991). We have extended our studies to examine the following: (i) transposition of Tn5 from sites in the phoA, phoB, proC, trpD, and ilvD genes; (ii) the effect of gene transcription; (iii) the comparative effect of dinD+ and dinD(Def) alleles; (iv) the use of a mating-out assay of transposition; (v) the effect of a recA(Prtc) allele located at the normal chromosomal site; and (vi) the effect at 41.5 degrees C of the recA441(Prtc) allele. The new results fully confirm our previous conclusions, including the fact that the frequency of Tn5 transposition under constitutive SOS conditions is site dependent.