Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis.

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica.

The industrial yeast Yarrowia lipolytica secretes high amounts of an alkaline extracellular protease encoded by the XPR2 gene. The industrial use of the XPR2 promoter was however hindered by its complex regulation. We designed hybrid promoters, based on tandem copies of the XPR2 promoter UAS1 region. In contrast to native XPR2 promoter, these hybrid promoters were not repressed by the preferred carbon and nitrogen sources, nor by acidic conditions, and they did not require the presence of peptones in the culture medium. They exhibited a strong quasi-constitutive activity, similar when carried on either integrative or replicative plasmids. We used these hybrid promoters to direct the production of bovine prochymosin, using XPR2 secretion signals. The production of active chymosin was several fold higher than with previously available Y. lipolytica promoters (up to 160 mg/l). Integrative vectors based on the hybrid promoters, allowing the easy insertion of a heterologous gene and its expression or expression/secretion in Y. lipolytica, were designed. We also designed new Y. lipolytica recipient strains with good secreting abilities, able to grow on sucrose, and devoid of extracellular proteases. These new tools will add to the interest of Y. lipolytica as a host for heterologous protein production.

Use of an infectious Simian virus 40-based shuttle vector to analyse UV-induced mutagenesis in monkey cells.

SV40 based shuttle vectors able to be packaged as pseudovirions have been used either as naked DNA or as pseudovirus to analyse the mutation frequency and the UV-induced mutation spectra obtained after transfection or infection of COS7 monkey cells. The frequency of supF spontaneous mutants was similar whatever the state of the vector, indicating that the transfection step is not responsible for the high spontaneous mutation frequency when using shuttle vectors. Nevertheless the UV-induced mutation frequency of the supF gene was higher when transfected DNA was replicated into COS7 cells than when pseudovirus infection was performed. The UV induced mutation spectra was basically similar in both situations but a new hot-spot at nucleotide 110 was obtained after pseudovirus infection. UV-pretreated and control COS7 cells were infected with untreated or UV-damaged pi SVPC7 shuttle virus and the survival and the supF mutation frequency were analysed in the progeny. The survival of UV-damaged pseudovirus replicated in 10 J/m2 UV-pretreated cells was 2-fold higher than in untreated cells. This increase in the survival was accompanied by a slight enhancement in the number of supF mutants.

Mutation spectrum of 4-nitroquinoline 1-oxide-damaged single-stranded shuttle vector DNA transfected into monkey cells.

4-Nitroquinoline 1-oxide (4NQO) is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. We recently determined the mutation spectrum induced by the ultimate metabolite of 4NQO, acetoxy-4-aminoquinolone 1-oxide in the M13lacZ'/E. coli lacZ delta M15 alpha-complementation assay. Our data suggested that dGuo-C8-AQO induces (per se or via AP sites) G to Pyr transversions. Here we report our study on 4NQO mutagenesis in monkey cells. 4NQO lesions were induced in vitro on a single-stranded (ss) DNA shuttle vector carrying the supF tRNA gene. This vector was able to replicate both in mammalian cells and in bacteria. The mutations induced in monkey cells were screened by the white/blue beta-galactosidase activity assay in E. coli. We took advantage of the peculiar feature of ss supF DNA in which the extent of secondary structure may be a function of the temperature, with the dependence of the 4NQO-specific adduct spectrum on DNA secondary structure. We reasoned that mutational spectra derived from damage induced in the presence (20 degrees C) or absence (70 degrees C) of DNA secondary structure should be different. The result of sequencing a total of 89 induced and spontaneous mutants confirmed that the spectra are statistically different. These data suggest that the two 4NQO guanine adducts may induce different mutations.

UV-induced mutations in a shuttle vector replicated in repair deficient trichothiodystrophy cells differ with those in genetically-related cancer prone xeroderma pigmentosum.

Trichothiodystrophy (TTD) is a rare genetic disease associated in approximately 50% of patients with DNA repair deficiency analogous to that found in xeroderma pigmentosum group D (XP-D) patients. Although XP-D patients exhibit a very high level of skin cancer on sun-exposed parts, TTD is not associated with cancer. We analysed UV-induced mutations in TTD cells and compared them to data in XP-D in order to determine if the molecular mechanisms of mutagenesis can explain the discrepancies between these two syndromes. We first immortalized a fibroblast TTD line with an ori(-)-SV40 plasmid. To investigate the kinds of mutations induced in TTD cells, we used an UV-irradiated (at 254 nm) shuttle vector carrying the supF tRNA gene as a target. We compared our data with those published by others with the same pZ189 vector in normal and XP-D fibroblast lines (Bredberg et al., Proc. Natl. Acad. Sci. USA, 83, 8273-8277; Seetharam et al., J. Clin. Invest., 80, 1613-1617). The frequency of mutants increased linearly with UV dose and the slope was > 4 times steeper in TTD cells than that observed in normal cells. The mutation frequency was almost identical between XP-D and TTD cells. Sequence analysis of the supF tRNA gene showed that 96% of mutations obtained in TTD cells are base substitutions. Single base substitutions were found in 62% of mutants in TTD cells while they corresponded to 86% in XP-D cells. The frequency of multiple mutations in TTD cells (26%) was similar to that in normal cells (27%) and much higher than that in XP-D cells (9%). Despite the fact that the same gene is mutated in TTD and XP patients, the molecular characteristics of mutagenesis are not identical. The fact that the frequency of mutations in TTD and XP cells are similar shows that a high level of UV-induced mutations is therefore not always directly related to cancer-proneness. Other factors such as catalase activity and immuno-surveillance may intervene in cancer incidence.

Single stranded DNA-vectors for analyzing processing of DNA damage induced by 4-nitroquinoline-1-oxide in prokaryotes and eukaryotes.

Previous studies indicate that single stranded DNA vectors could be used in different organisms to study mutagenesis induced by DNA damaging agents. We applied this approach to study mutagenesis induced by 4NQO lesions. The use of ssDNA, on which the ultimate metabolite of 4NQO (Ac-4HAQO) induces mainly C8-guanine adducts, allowed us to find a correlation between G-transversions and the dGuo-C8-AQO adduct. This correlation was established in two independent assay-systems, based on prokaryotic and eukaryotic cells.

Spontaneous and ultraviolet-induced mutations on a single-stranded shuttle vector transfected into monkey cells.

The shuttle vector plasmid PCF3A, carrying the supF target gene, can be transfected into monkey COS7 cells as single-stranded or double-stranded DNA. Single strand-derived plasmid progeny exhibited a 10-fold higher spontaneous mutation frequency than double strand-derived progeny. The location of spontaneous mutations obtained after transfection of the single-stranded vector shared similarities with that for double-stranded vectors. However, the nature of base changes was very different. Single-stranded PCF3A DNA was used to study ultraviolet-induced mutagenesis. An earlier report (Madzak and Sarasin, J. Mol. Biol., 218 (1991) 667-673) showed that single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after ultraviolet irradiation. In the present report, sequence analysis of mutant plasmids is presented. The use of a single-stranded vector allowed us to show the targeting of mutations at putative lesion sites and to determine the exact nature of the base implicated in each mutation. Frameshift mutations were more frequent after transfection of control or irradiated plasmid as single-stranded DNA than as double-stranded DNA. Multiple mutations, observed at a high frequency in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, could be due to an error-prone polymerisation step acting on a single-stranded template.

Efficient in vivo encapsidation of a shuttle vector into pseudo-simian virus 40 virions using a shuttle virus as helper.

We have designed shuttle vectors containing the late region of simian virus 40 (SV40) DNA (coding for the capsid proteins) which could be encapsidated into pseudo-SV40 virions during passage in monkey cells. We describe here the use of these shuttle viruses as helpers for the encapsidation of another shuttle vector into viral particles. Following cotransfection into monkey cells, the efficiency of encapsidation was similar for the shuttle virus and the other plasmid. The amounts of pseudo-SV40 virions recovered from the two vectors reflected the amounts of their DNA present in monkey cells. Thus, the presence of the SV40 late region did not confer any significant advantage for encapsidation. The encapsidation of any shuttle vector into pseudo-SV40 virions is therefore possible and efficient, shuttle viruses constituting an interesting alternative to the use of SV40 as helper in this process.

Singlet oxygen induced DNA damage and mutagenicity in a single-stranded SV40-based shuttle vector.

The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2 (endoperoxide of the disodium 3,3'-(1,4-naphthylidene) dipropionate), on a single-stranded shuttle vector were analysed. 1O2 induces a much higher level of breaks in the phosphodiester backbone of single-stranded than double-stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by 1O2. The damaged vector was transfected into monkey COS7 cells where single-stranded DNA was converted to the double-stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued in E. coli to study mutagenesis. There is a significant increase in mutation frequency of damaged single-stranded DNA in comparison to untreated DNA. It is concluded that 1O2 induces breaks in the backbone of single-stranded DNA and that the 1O2-damaged molecules are mutated after passage through mammalian cells.

Conversion of a single-stranded simian virus 40 (SV40)-based shuttle vector to its double-stranded form does not require the SV40 T antigen in monkey cells.

We have transfected the single-stranded DNA form of the simian virus 40 (SV40)-based shuttle vector pZ189 into CV1P simian cells. Although the strand used did not code for the T antigen, we observed its conversion to a double-stranded DNA form. We deduced that the replication step converting a circular single-stranded DNA to a double-stranded one is independent of the SV40 T antigen in simian cells.

Mutation spectrum following transfection of ultraviolet-irradiated single-stranded or double-stranded shuttle vector DNA into monkey cells.

We designed a shuttle vector system that allowed a comparison of the mutation spectrum on the supF target gene after transfection of single-stranded or double-stranded DNA into monkey cells. Single-strand-derived plasmids exhibited a spontaneous mutation frequency tenfold higher than double-strand-derived ones. These spontaneous mutations comprised deletions and point substitutions. This system was applied to the study of ultraviolet-induced mutagenesis. Single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after identical ultraviolet-irradiation. The use of single-stranded DNA allowed us to confirm and complete the data about the targeting of ultraviolet-induced mutations and the exact nature of the base changes involved. One class of mutations was more frequent after transfection of ultraviolet-irradiated single-stranded DNA than for double-stranded DNA: frameshifts represented 10% of the mutants. Multiple mutations, attributed by some authors to an error-prone excision repair process, have also been observed in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, although it cannot be a direct substrate for excision repair.

Mutagenic properties of a unique abasic site in mammalian cells.

The mutagenic properties of a true unique abasic site located opposite a guanine residue were studied. An oligonucleotide containing a chemically-produced abasic site was inserted into a shuttle vector able to replicate both in simian cells and in bacteria. Plasmid DNA was rescued from simian cells and screened in bacteria by differential hybridization with a labelled oligonucleotide probe. Mutations were easily detected and sequenced. Results showed that opposite a guanine the abasic site was error free repaired or replicated by mammalian cells with an efficiency of 99%. Point mutations occurred at a frequency of approximately 1% in control host cells and at more than 3% in UV-pre-irradiated host cells. Adenine, cytosine or thymine were found to have been inserted opposite the abasic site. No preferential insertion for a particular base was observed in contrast to that reported in bacteria.

Mechanisms and consequences of mutation induction in mammalian cells.

Mutations have been studied for several decades in order to understand biological processes of great significance and the selection of better-adapted species. Our knowledge both of mutation spectra induced by genotoxic agents and the mechanisms involved in DNA damage processing is more advanced in bacteria than in animal cells. However, the use of new technologies such as shuttle vectors or the polymerase chain reaction will undoubtedly allow rapid progress in the next few years. Shuttle vectors consist of target sequences for monitoring mutagenic activity and additional sequences permitting DNA replication and selection, both in bacteria and in mammalian cells. These plasmids are very efficient in allowing the production of mutation spectra of a particular genotoxin in animal cells. In most cases, base substitutions occur predominantly at the sites of base damage and the type of substitution depends on the kind of damage. This has been well characterized using ultraviolet (UV) light as a mutagen. UV-induced mutations are targeted opposite pyrimidine-pyrimidine sites, where the two major UV lesions are produced. The direct relationships existing between mutation and cancer are exemplified by some hereditary diseases where deficiency in an enzymatic repair system is linked to a high incidence of tumours. Similarly, activation of some cellular proto-oncogenes occurs via specific point mutations. A correlation does exist between the mutation spectra found in model systems and the specific mutation found in the activated oncogene in tumours induced by a given genotoxin. This is particularly well illustrated in the DNA repair deficiency syndrome, xeroderma pigmentosum. The specific mutations found in activated ras oncogenes isolated from UV-stimulated skin tumours correlate well with the mutagenic properties of unrepaired UV-induced DNA lesions.

SV40-based shuttle viruses.

We summarize in this paper the advantages of the shuttle virus system. These SV40-based vectors exhibit the unique properties of being packaged as SV40 pseudo-virions and of being able to infect host cells. Using these transient vectors, we show that their replication can be regulated in some monkey cell lines, in such a way that either low or very high amounts of plasmid DNA can be obtained. The stability of these infectious shuttle vectors in different conditions is analyzed by rescuing them in E. coli, using various gene mutation targets. Moreover, we describe a new series of vectors which can be produced as single-stranded DNA in bacteria. They allow the transfection of a plasmid genome into mammalian cells, as either single-stranded or double-stranded DNA.

Analysis of single-stranded DNA stability and damage-induced strand loss in mammalian cells using SV40-based shuttle vectors.

The fate and stability of fully or partially single-stranded DNA molecules transfected into mammalian cells have been analysed. For this, we constructed a simian virus 40 (SV40)-based shuttle vector containing the f1 bacteriophage replication origin in the two possible orientations (pi SVF1-A and pi SVF1-B). This vector contains the SV40 origin of replication, the late viral genes and DNA sequences for replication and selection in Escherichia coli. It also carries the lacO sequence, which permits the analysis of plasmid stability. Single-stranded DNA from pi SVF1-A and pi SVF1-B were produced in bacteria and annealed in vitro to form a heteroduplex molecule. We showed that, in monkey kidney COS7 cells, single-stranded vectors replicate to form duplex molecules. After transfection of the three forms of molecules (single-stranded, heteroduplex or double-stranded), replicated DNA was rescued in E. coli. Vector stability was analysed by checking for plasmid rearrangements and screening for lacO mutants. The single-stranded pi SVF1 has a lower rearrangement level, while the spontaneous mutation frequency (on the lacO target) is in the same range as for the double-stranded vector. In contrast, the level of spontaneous mutagenesis is higher for the heteroduplex than for the single- and double-stranded forms. In addition, we found that replication of heteroduplex with one strand containing ultraviolet light-induced lesions yields progeny molecules in which the irradiated strand is mostly lost. This result indicates for the first time the specific loss of the damaged strand in mammalian cells.