The two-step model of UV mutagenesis reassessed: deamination of cytosine in cyclobutane dimers as the likely source of the mutations associated with photoreactivation

Summary A large increase in the incidence of bacteriophage mutants is found after photoreactivation of UV-irradiated phage S13. The increase was seen only when the irradiated phage were stored before they were photoreactivated; the maximum mutation frequency was achieved after storage for 2 h at 4° C or 30 min at 37° C. The mutations can be attributed entirely to deamination of cytosine in cyclobutane dimers. Naked S13 DNA was stored for 2 h at 37° C after being irradiated with wavelengths ≥290 nm in the presence of 0.2% acetophenone, which sensitizes the formation of thymine-thymine but not cytosine-containing dimers; the specific mutation frequency was 7.2-fold lower compared to the frequency produced by irradiation in the absence of the photosensitizer, confirming that cytosine dimers are a major source of mutations. These results undermine the basis for the two-step model of UV mutagenesis in which a distinctly separate misincorporation step is supposed to precede the lesion bypass step; instead the results support a different two-step model, in which a deamination step precedes the bypass. The S13 capsid appears to completely inhibit the putative deamination reaction at about 75% of the dimer sites.     

Polymerases and UV mutagenesis in Escherichia coli

Evidence for and against the involvement of the known nucleic acid polymerases in UV mutagenesis in Escherichia coli is reviewed. There is no evidence that rules out the participation of any of them when they are present but only one, the alpha subunit of DNA polymerase III holoenzyme (polC gene product) has been shown to be essential. It is argued that the PolC protein that functions in UV mutagenesis may not be immediately recognizable as one of the normal cellular polymerases or polymerase complexes.     

紫外诱变选育恩拉霉素高产菌株

本研究采用紫外诱变育种技术对一株产恩拉霉素抗真菌链霉菌(Streptomyces fungicidicus)F110进行了诱变处理,经链霉素抗性、利福霉素B抗性以及双重抗性筛选,共获得了132株抗生素抗性突变株,其中26株突变菌株的恩拉霉素产量与出发菌株相比均有明显提高。摇瓶发酵条件下,突变株SR93的恩拉霉素产量最高可达2 400μg/m L,与出发菌株相比提高了38%。传代结果表明:该突变株产素水平稳定,因此具备较好的开发及工业应用价值。     

Chemical and UV mutagenesis in Zymomonas mobilis

Mutagenesis of the facultative anaerobe Zymomonas mobilis was accomplished by three different mutagens. Ultra-violet (UV) irradiation, whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, was a poor mutagen for this organism. Ethyl methane sulphonate (EMS) gave results very similar to UV-irradiation. N-methyl-N-nitro-N-nitrosoguanidine (MNNG), which is believed to act by multiple mutagenic mechanisms, was the most powerful mutagen, always resulting in a large number of mutants of all types examined (i.e. auxotrophs, antibiotic resistant, heavy metal resistant and ultraviolet sensitive). Reversion frequencies of MNNG-induced mutants were very low. Evidence is provided that mutagenesis of Z. mobilis is affected by photoreactivation, adaptive response and error-prone repair mechanisms. Moreover, cells treated with alkylating agents and allowed to recover under anaerobic conditions clearly demonstrated that anaerobiosis plays a significant role in repair, but not in the induction of mutants.     

The role of nutrient broth supplementation in UV mutagenesis of E. coli

Postirradiation expression of UV-induced reversion is examined to understand the different yields of E. coli revertants on agar media unsupplemented, supplemented with a small amount of the required amino acid, or supplemented with nutrient broth. Protein synthesis is determined in irradiated cells incubating on these three media by observing the incorporation of [³H]proline. Nutrient broth supplementation is known to yield a large number of suppresor-containing revertants which does not develop with supplementation of a small amount of the required amino acid. However, the rates of protein synthesis in cells on these two media are found to be similar. Consequently the rate of protein synthesis per se does not seem responsible for enhance expression of suppressor-containing revertants. An empirical model is proposed to realte nutrient broth enhancement to mutation frequency decline and finalization of GC to AT transitions.     

UV mutagenesis in inhibitor depleted conidia of Aspergillus nidulans

UV treated conidia of a strain of Aspergillus nidulans (meth Gl. biAl) depleted of germination inhibitory substances have been examined for inactivation and mutation induction at groups of suppressor gene loci defining three classes of methionine revertants. An exponential decline in the colony forming ability and quadratic increase in mutation frequency (for each class of revertant) as the incident dose increased were observed. The induced mutation frequency for each class and the loss of colony forming ability of the conidia are greater in the absence than in the presence of these self-inhibitors of germination. However, the relative frequencies of the individual classes of revertants did not differ whether the inhibitory substances were present or not. Liquid holding effects leading to increases in survival and mutation have been observed, but the relative frequencies of the individual revertant classes remain unchanged.     

UV and chemical mutagenesis in rev7 mutants of yeast

Summary We have examined induced mutagenesis in rev7-1 mutants of Baker's yeast' Saccharomyces cerevisiae, using a variety of contrasting test systems and several different mutagens. UV-induced reversion frequencies of the ochre allele arg4-17, the putative missense allele ilv1-92 and the frameshift allele his4-38 were 10 to 200 fold lower in haploid and diploid rev7-1 mutants compared with wild type strains, but UV-induced reversion frequencies of the frameshift allele leu2-3 and the proline missense allele cyc1-115 were reduced only a few fold. Ilv1-92 reversion frequencies induced by methyl methane sulfonate or by N-methyl-N-nitro-N-nitrosoguanidine were 10 to 20 times lower in rev7-1 mutants, but normal frequencies of these revertants were induced with ethyl methane sulfonate, even though rev7-1 strains are slightly sensitive to this mutagen as well as to the others tested. We conclude that the rev7 mutants, like the rev3 mutants they closely resemble, have a substantial but not total deficiency concerning induced mutagenesis.     

The two-step model for translesion synthesis: then and now

The formation of base substitution mutations following exposure of bacteria to ultraviolet light and many other mutagens occurs during translesion synthesis opposite a photoproduct or other lesion in the template strand of DNA. This process requires the UmuD(2)' UmuC complex, only formed to a significant extent in SOS-induced cells. The "two-step" model proposed that there were two steps, insertion of a wrong base (misincorporation) and use of the misincorporated base as a primer for further chain extension (bypass). The original evidence suggested that UmuD(2)' UmuC was needed only for the second step and that in its absence other polymerases such as DNA polymerase III could make misincorporations. Now we know that the UmuD(2)' UmuC complex is DNA polymerase V and that it can carry out both steps in vitro and probably does both in vivo in wild-type cells. Even so, DNA polymerase III clearly has an important accessory role in vitro and a possibly essential role in vivo, the precise nature of which is not clear. DNA polymerases II and IV are also up-regulated in SOS-induced cells and their involvement in the broader picture of translesion synthesis is only now beginning to emerge. It is suggested that we need to think of the chromosomal replication factory as a structure through which the DNA passes and within which as many as five DNA polymerases may need to act. Protein-protein interactions may result in a cassette system in which the most appropriate polymerase can be engaged with the DNA at any given time. The original two-step model was very specific, and thus an oversimplification. As a general concept, however, it reflects reality and has been demonstrated in experiments with eukaryotic DNA polymerases in vitro.     

sTarPicker: a method for efficient prediction of bacterial sRNA targets based on a two-step model for hybridization

Background

Bacterial sRNAs are a class of small regulatory RNAs involved in regulation of expression of a variety of genes. Most sRNAs act in trans via base-pairing with target mRNAs, leading to repression or activation of translation or mRNA degradation. To date, more than 1,000 sRNAs have been identified. However, direct targets have been identified for only approximately 50 of these sRNAs. Computational predictions can provide candidates for target validation, thereby increasing the speed of sRNA target identification. Although several methods have been developed, target prediction for bacterial sRNAs remains challenging.

Results

Here, we propose a novel method for sRNA target prediction, termed sTarPicker, which was based on a two-step model for hybridization between an sRNA and an mRNA target. This method first selects stable duplexes after screening all possible duplexes between the sRNA and the potential mRNA target. Next, hybridization between the sRNA and the target is extended to span the entire binding site. Finally, quantitative predictions are produced with an ensemble classifier generated using machine-learning methods. In calculations to determine the hybridization energies of seed regions and binding regions, both thermodynamic stability and site accessibility of the sRNAs and targets were considered. Comparisons with the existing methods showed that sTarPicker performed best in both performance of target prediction and accuracy of the predicted binding sites.

Conclusions

sTarPicker can predict bacterial sRNA targets with higher efficiency and determine the exact locations of the interactions with a higher accuracy than competing programs. sTarPicker is available at http://ccb.bmi.ac.cn/starpicker/.     

N-nitrosamines: bacterial mutagenesis and in vitro metabolism

Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to alpha-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., alpha-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 X g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolution of the two-step model for UV-mutagenesis

It is quite remarkable how our understanding of translesion DNA synthesis (TLS) has changed so dramatically in the past 2 years. Until very recently, little was known about the molecular mechanisms of TLS in higher eukaryotes and what we did know, was largely based upon Escherichia coli and Saccharomyces cerevisiae model systems. The paradigm, proposed by Bryn Bridges and I [Mutat. Res. 150 (1985) 133] in 1985, was that error-prone TLS occurred in two steps; namely a misinsertion event opposite a lesion, followed by extension of the mispair so as to facilitate complete bypass of the lesion. The initial concept was that at least for E. coli, the misinsertion event was performed by the cell's main replicase, DNA polymerase III holoenzyme, and that elongation was achieved through the actions of specialized polymerase accessory proteins, such as UmuD and UmuC. Some 15 years later, we now know that this view is likely to be incorrect in that both misinsertion and bypass are performed by the Umu proteins (now called pol V). As pol V is normally a distributive enzyme, pol III may only be required to "fix" the misincorporation as a mutation by completing chromosome duplication. However, while the role of the E. coli proteins involved in TLS have changed, the initial concept of misincorporation followed by extension/bypass remains valid. Indeed, recent evidence suggests that it can equally be applied to TLS in eukaryotic cells where there are many more DNA polymerases to choose from. The aim of this review is, therefore, to provide a historical perspective to the "two-step" model for UV-mutagenesis, how it has recently evolved, and in particular, to highlight the seminal contributions made to it by Bryn Bridges.     

An approach to analyzing UV mutagenesis in E. coli

UV mutagenesis of single-strand DNA phage can be divided into three types: induced untargeted; induced targeted; and uninduced targeted. We report the development of new tools to determine the number of processes which contribute to these types of mutagenesis. An E. coli tRNA gene, glyU, has been cloned using M13 derivatives mp8 and mp9 as vectors. The nucleotide sequence of glyU and its flanking regions is presented. In this paper, phage glyU anticodon mutants are detected by their ability to suppress GAA and GAT missense mutations in trpA. We used phage carrying GAG and CTC at the anticodon position and found results consistent with the hypothesis that two processes act to produce the transition to GAA suppression: an uninduced regionally targeted process; and an induced locally targeted process with some untargeted activity. The transversion frequency to GAT suppression on the other hand responded as if only an uninduced locally targeted process was involved. Thus, we hypothesize that the new tools have discriminated three different processes of mutagenesis and we discuss further work designed to test this hypothesis.