Development of a thermostable Cre/lox-based gene disruption system and in vivo manipulations of the megaplasmid pTT27 in Thermus thermophilus HB27.

We previously reported the development of a Cre/lox-based gene disruption system for multiple markerless gene disruption in Thermus thermophilus; however, it was a time-consuming method because it functioned at 50 °C, the minimum growth temperature of T. thermophilus HB27. In the present study, we improved this system by introducing random mutations into the cre-expressing plasmid, pSH-Cre. One of the resulting mutant plasmids, pSH-CreFM allowed us to remove selection marker genes by Cre-mediated recombination at temperatures up to 70 °C. By using the thermostable Cre/lox system with pSH-CreFM, we successfully constructed two valuable pTT27 megaplasmid mutant strains, a plasmid-free strain and ß-galactosidase gene deletion strain, which were produced by different methods. The thermostable Cre/lox system improved the time-consuming nature of the original Cre/lox system, but it was not suitable for multiple markerless gene disruption in T. thermophilus because of its highly efficient induction of Cre-mediated recombination even at 70 °C. However, in vivo megaplasmid manipulations performed at 65 °C were faster and easier than with the original Cre/lox system. Collectively, these results indicate that this system is a powerful tool for engineering T. thermophilus megaplasmids.

Detection and analysis of UV-induced mutations in the chromosomal DNA of Arabidopsis.

Under natural conditions, plants are exposed to solar ultraviolet (UV) radiation, which damages chromosomal DNA. Although plant responses to UV-induced DNA damage have recently been elucidated in detail, revealing a set of DNA repair mechanisms and translesion synthesis (TLS), limited information is currently available on UV-induced mutations in plants. We previously reported the development of a supF-based system for the detection of a broad spectrum of mutations in the chromosomal DNA of Arabidopsis. In the present study, we used this system to investigate UV-induced mutations in plants. The irradiation of supF-transgenic plants with UV-C (500 and 1000 J/m2) significantly increased mutation frequencies (26- and 45-fold, respectively). G:C to A:T transitions (43-67% of base substitutions) dominated in the mutation spectrum and were distributed throughout single, tandem, and multiple base substitutions. Most of these mutations became undetectable with the subsequent illumination of UV-irradiated plants with white light for photoreactivation (PR). These results indicated that not only G:C to A:T single base substitutions, but also tandem and multiple base substitutions were caused by two major UV-induced photoproducts, cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). In contrast, a high proportion of A:T to T:A transversions (56% of base substitutions) was a characteristic feature of the mutation spectrum obtained from photoreactivated plants. These results define the presence of the characteristic feature of UV-induced mutations, and provide insights into DNA repair mechanisms in plants.

Development of a supF-based mutation-detection system in the extreme thermophile Thermus thermophilus HB27.

Thermus thermophilus (T. thermophilus) HB27 is an extreme thermophile that grows optimally at 65-72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of T. thermophilus than in those of mesophiles; however, the mechanisms underlying the maintenance of genome integrity at high temperatures remain poorly understood. The study of mutation spectra has become a powerful approach to understanding the molecular mechanisms responsible for DNA repair and mutagenesis in mesophilic species. Therefore, we developed a supF-based system to detect a broad spectrum of mutations in T. thermophilus. This system was validated by measuring spontaneous mutations in the wild type and a udgA, B double mutant deficient in uracil-DNA glycosylase (UDG) activity. We found that the mutation frequency of the udgA, B strain was 4.7-fold higher than that of the wild type and G:C→A:T transitions dominated, which was the most reasonable for the mutator phenotype associated with the loss of UDG function in T. thermophilus. These results show that this system allowed for the rapid analysis of mutations in T. thermophilus, and may be useful for studying the molecular mechanisms responsible for DNA repair and mutagenesis in this extreme thermophile.

Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus.

Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP-htk-loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination. We found that the Cre/lox system was compatible with the proliferation of the T. thermophilus HB27 strain at the lowest growth temperature (50 °C), and thus succeeded in establishing a triple gene disruptant, the (∆TTC1454::loxP, ∆TTC1535KpnI::loxP, ∆TTC1576::loxP) strain, without leaving behind a selectable marker. During the process of the sequential disruption of multiple genes, we observed the undesired deletion and inversion of the chromosomal region between multiple loxP sites that were induced by Cre-mediated recombination. Therefore, we examined the effects of a lox66-htk-lox71 cassette by exploiting the mutant lox sites, lox66 and lox71, instead of native loxP sites. We successfully constructed a (∆TTC1535::lox72, ∆TTC1537::lox72) double gene disruptant without inducing the undesired deletion of the 0.7-kbp region between the two directly oriented lox72 sites created by the Cre-mediated recombination of the lox66-htk-lox71 cassette. This is the first demonstration of a Cre/lox system being applicable to extreme thermophiles in a genetic manipulation. Our results indicate that this system is a powerful tool for multiple markerless gene disruption in T. thermophilus.

Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis.

The factors maintaining genomic integrity, which have been studied in detail in other species, have yet to be investigated in plants. Recent progress in gene-silencing technology has made it possible to produce transgenic plants with loss-of-function phenotypes for the effective analysis of these factors, even with the high redundancy of genes in plants. Therefore, a mutation-detection system for plants is necessary to estimate the biological function of a target gene for mutation frequencies and spectra. Here, we reported the development of a novel system to analyze mutations in the chromosomal DNA of plants. The supF gene of E. coli was used as a target for the mutation because it was possible to detect all mutational base changes. Based on the plasmid pTN30, which carries supF, we constructed a binary Ti vector for its introduction to Arabidopsis genomes. The system was validated by measuring mutations in both non-treated and mutagen-treated transgenic plants. DNA fragments including pTN30 were rescued from the plants, and introduced into E. coli KS40/pOF105 to isolate the supF mutant clones conferring both nalidixic acid and streptomycin resistance on transformants. We found that the mutation frequency was approximately three times higher with the ethyl methanesulfonate (EMS) treatment than without it and G:C to A:T transitions dominated, which was the most reasonable mutation induced by EMS. These results show that this system allowed for the rapid analysis of mutations in plants, and may be useful for analyzing plant genes related to the functions of genomic stability and monitoring environmental genotoxic substances.

Phenyl hydroquinone, an Ames test-negative carcinogen, induces Hog1-dependent stress response signaling.

Recently, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae. We further found that phenyl hydroquinone arrested the cell cycle at G(1) and G(2)/M. In this study, we demonstrate that phenyl hydroquinone can arrest the cell cycle at the G(2)/M transition as a result of stabilization of Swe1 (a Wee1 homolog), probably leading to inactivation of Cdc28 (a Cdk1/Cdc2 homolog). Furthermore, Hog1 (a p38 MAPK homolog) was robustly phosphorylated by phenyl hydroquinone, which can stabilize Swe1. On the other hand, Chk1 and Rad53 were not phosphorylated by phenyl hydroquinone, indicating that the Mec1/Tel1 DNA-damage checkpoint was not functional. Mutations of swe1 and hog1 abolished phenyl hydroquinone-induced arrest at the G(2)/M transition and the cells became resistant to phenyl hydroquinone lethality and aneuploidy development. These data suggest that a phenyl hydroquinone-induced G(2)/M transition checkpoint that is activated by the Hog1-Swe1 pathway plays a role in the development of aneuploidy.

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides.

Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with H2O2, and the rpoB mutant frequencies were calculated. The frequency of the H2O2-induced mutations was increased in all of the strains tested; however, the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.

Ames test-negative carcinogen, ortho-phenyl phenol, binds tubulin and causes aneuploidy in budding yeast.

Ortho-phenyl phenol (OPP) is broad-spectrum of fungicides and antibacterial agents. OPP tested negative in an Ames system and positive with respect to the formation of tumors in the urinary bladder in rats when administered in diet, showing attributes of an Ames test-negative carcinogen. It has also been demonstrated that OPP does not bind or cleave DNA in vivo or in vitro, rather dose-dependent protein binding in OPP-treated rats was observed. OPP, however, generates chromosomal aberrations including aneuploidy. Thus, the steps by which Ames test-negative carcinogens exert their effects need to be elucidated. Here, we used an assay of loss of heterozygosity (LOH) in Saccharomyces cerevisiae to determine the biological effects of OPP and its hepatic metabolite phenyl hydroquinone (PHQ). LOH was found to be induced by OPP and PHQ because of a functional chromosome loss: aneuploidy. PHQ bound to and interfered with the depolymerization of tubulin in vitro and arrested the cell-cycle at M and G1. These results indicate that OPP and PHQ damaged tubulin to cause mis-segregation of chromosome by delaying cell-cycle progression through mitosis, and as a consequence caused aneuploidy.

Involvement of Y-family DNA polymerases in mutagenesis caused by oxidized nucleotides in Escherichia coli.

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.

Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae.

To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.

A novel Nudix hydrolase for oxidized purine nucleoside triphosphates encoded by ORFYLR151c (PCD1 gene) in Saccharomyces cerevisiae.

A search for candidates for a functional homologue of Escherichia coli MutT in yeast Saccharomyces cerevisiae was made in the NCBI-BLAST database using the Nudix box, a short amino acid sequence conserved among E.coli MutT, Pseudomonoas vulgaris MutT, and human, rat and mouse MTH1. Among five candidates, we focused on the open reading frame YLR151c, because it had a region with approximately 76% similarity to the N-terminal half of MutT including the Nudix box. We thus evaluated the ability of YLR151c as a functional homologue of E.coli MutT in S.cerevisiae. Expression of YLR151c was able to suppress the transversion from A:T to C:G caused by misincorporation of the oxidized nucleotide 8-oxo-dGTP in the E.coli mutT-deficient strain. The disruption of the YLR151c in yeast strain caused approximately 14-fold increase in the frequency of spontaneous mutation compared to the wild type. Additionally, biochemical analysis indicated that GST-YLR151c fusion protein possessed pyrophosphatase activity for both 7,8-dihydro-8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP) and 1,2-dihydro-2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP). The specific activity of GST-YLR151c for 8-oxo-dGTP was 5.6 x 10(-3) microM(-1) s(-1), which was similar to that of RibA, a backup enzyme for MutT in E.coli, but was 150-fold lower than that of hMTH1. From these results, we conclude that YLR151c has an ability to prevent spontaneous mutagenesis via sanitization of oxidized nucleotides, and that it may be the functional homologue of E.coli MutT in S.cerevisiae.

Hydrogen peroxide-induced microsatellite instability in the Escherichia coli K-12 endogenous tonB gene.

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using an endogenous tonB gene as a mutation selective marker in Escherichia coli, we have examined whether endogenous oxidative mutagenesis can contribute to genetic instability. We have also used oxyR(+) and oxyR(-) strains to evaluate how hydrogen peroxide scavenging system can contribute to genetic instability. The highest mutation frequency induced by hydrogen peroxide was 3.8x10(-6) at 600 microM and 5.3 x 10(-6) at 40 microM in oxyR(+) and oxyR(-), respectively. Hydrogen peroxide induced minus frameshift mutations predominantly followed by transversions (G:C-->T:A, G:C-->C:G, and A:T-->T:A). The types and the nature of the mutations did not differ between strains. Frameshift mutations occurred at G:C and A:T sites equally, and in repeated and non-repeated sequences equally. It is evident that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication and contribute to genomic instability. Our results further indicate that oxyR regulon does not take part in the DNA-repair pathway against oxidative damage induced by hydrogen peroxide.

Characterization of spontaneous mutation in the delta soxR and SoxS overproducing strains of Escherichia coli.

To examine the role of the soxRS regulon in mutagenesis, we characterized the spontaneous mutations occurring in the endogenous tonB gene in the delta soxR strain and the SoxS overproducing strain of Escherichia coli. Neither the delta soxR strain nor the SoxS overproducing strain led to an enhancement or diminishment of the spontaneous mutation frequency. By DNA sequencing, we determined 50 spontaneous mutants from the delta soxR strains, and found that 36% were both base substitutions and IS insertions, 14% frameshifts and 10% deletions. Among the base substitutions, G:C-->T:A transversions and G:C-->A:T transitions predominated, followed by A:T-->T:A transversions. We determined 54 spontaneous mutants from the SoxS overproducing strains, and found that 37% were IS insertions, 31% base substitutions, 17% frameshifts, 9% deletions and 6% duplications. Among the base substitutions, G:C-->T:A transversions dominated, followed by A:T-->T:A transversions and G:C-->A:T transitions. These results were similar to those from the soxRS+ strains. Thus, it is suggested that the soxRS-regulated genes do not play a significant role in the defense against spontaneous mutagenesis.

Mutagenic target for hydroxyl radicals generated in Escherichia coli mutant deficient in Mn- and Fe- superoxide dismutases and Fur, a repressor for iron-uptake systems.

We previously reported that mutations in Mn- and Fe-superoxide dismutases and Fur, a repressor for iron uptake systems, simulated generation of hydroxyl radicals, and caused hypermutability in Escherichia coli. The predominant type of spontaneous mutation was GC --> TA, followed by AT --> CG, suggesting the involvement of 7,8-dihydro-8-oxoguanine (8-oxoG) and 1,2-dihydro-2-oxoadenine (2-oxoA) in DNA as well as 7,8-dihydro-8-oxodeoxyguanosine triphosphate (8-oxodGTP) and 1,2-dihydro-2-oxodeoxyadenosine triphosphate (2-oxodATP) in the nucleotide pool. To determine the targets contributing to oxidative mutagenesis, DNA or nucleotides, we characterized spontaneous mutations and compared the distribution to those in mutMY and mutT strains, in which GC --> TA and AT --> CG were predominantly induced, respectively. The hotspots and sequence contexts where AT --> CG occurred frequently in sodAB fur strain were almost identical to those in mutT strain,whereas, those where GC --> TA occurred frequently in sodAB fur strain were quite different from those in mutMY strain. These observations suggested that AT --> CG is due to 8-oxodGTP, while GC --> TA is produced by some other lesion(s). The 2-oxodATP is also a major oxidative lesion in nucleotides, and strongly induces GC --> TA. The expression of cDNA for MTH1, which can hydrolyze 2-oxodATP as well as 8-oxodGTP, partially but significantly, suppressed the GC --> TA mutator phenotype of the sodAB fur strain, whereas, it did not for the mutMY strain. Additionally, the sequence contextby 2-oxodATP in E. coli was similar to that in sodAB fur strain. These results suggested that the targets contributing to oxidative mutagenesis in sodAB fur strain are nucleotides such as dGTP and dATP, rather than DNA.