Adaptive mutation in Saccharomyces cerevisiae

Adaptive mutation is a generic term for processes that allow individual cells of nonproliferating cell populations to acquire advantageous mutations and thereby to overcome the strong selective pressure of proliferation-limiting environmental conditions. Prerequisites for an occurrence of adaptive mutation are that the selective conditions are nonlethal and that a restart of proliferation may be accomplished by some genetic change in principle. The importance of adaptive mutation is derived from the assumption that it may, on the one hand, result in an accelerated evolution of microorganisms and, on the other, in multicellular organisms may contribute to a breakout of somatic cells from negative growth regulation, i.e., to cancerogenesis. Most information on adaptive mutation in eukaryotes has been gained with the budding yeast Saccharomyces cerevisiae. This review focuses comprehensively on adaptive mutation in this organism and summarizes our current understanding of this issue.     

Adaptive Mutagenesis at ebgR Is Regulated by PhoPQ

Adaptive mutations are mutations that occur in nondividing or very slowly dividing microbial cells during prolonged nonlethal selection and that are specific to the challenge of the selection in the sense that the only mutations that can be detected are those that provide a growth advantage to the cell. The phoPQ genes encode a two-component positively acting regulatory system that controls expression of at least 25 to 30 genes in Escherichia coli and Salmonella typhimurium. PhoPQ responds to a variety of environmental stress signals including Mg²⁺ starvation and nutritional deprivation. Here I show that disruption of phoP or phoQ by Tn10dCam significantly reduces the adaptive mutation rate to ebgR, indicating that the adaptive mutagenesis machinery is regulated, directly or indirectly, by phoPQ. The finding that it is regulated implies that adaptive mutagenesis does not simply result from a failure of various error correction mechanisms during prolonged starvation.     

A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation

Special mechanisms of mutation are induced in microbes under growth-limiting stress causing genetic instability, including occasional adaptive mutations that may speed evolution. Both the mutation mechanisms and their control by stress have remained elusive. We provide evidence that the molecular basis for stress-induced mutagenesis in an E. coli model is error-prone DNA double-strand break repair (DSBR). I-SceI-endonuclease-induced DSBs strongly activate stress-induced mutations near the DSB, but not globally. The same proteins are required as for cells without induced DSBs: DSBR proteins, DinB-error-prone polymerase, and the RpoS starvation-stress-response regulator. Mutation is promoted by homology between cut and uncut DNA molecules, supporting a homology-mediated DSBR mechanism. DSBs also promote gene amplification. Finally, DSBs activate mutation only during stationary phase/starvation but will during exponential growth if RpoS is expressed. Our findings reveal an RpoS-controlled switch from high-fidelity to mutagenic DSBR under stress. This limits genetic instability both in time and to localized genome regions, potentially important evolutionary strategies.     

A mutation-promotive role of nucleotide excision repair in cell cycle-arrested cell populations following UV irradiation

Growing attention is paid to the concept that mutations arising in stationary, non-proliferating cell populations considerably contribute to evolution, aging, and pathogenesis. If such mutations are beneficial to the affected cell, in the sense of allowing a restart of proliferation, they are called adaptive mutations. In order to identify cellular processes responsible for adaptive mutagenesis in eukaryotes, we study frameshift mutations occurring during auxotrophy-caused cell cycle arrest in the model organism Saccharomyces cerevisiae. Previous work has shown that an exposure of cells to UV irradiation during prolonged cell cycle arrest resulted in an increased incidence of mutations. In the present work, we determined the influence of defects in the nucleotide excision repair (NER) pathway on the incidence of UV-induced adaptive mutations in stationary cells. The mutation frequency was decreased in Rad16-deficient cells and further decreased in Rad16/Rad26 double-deficient cells. A knockout of the RAD14 gene, the ortholog of the human XPA gene, even resulted in a nearly complete abolishment of UV-induced mutagenesis in cell cycle-arrested cells. Thus, the NER pathway, responsible for a normally accurate repair of UV-induced DNA damage, paradoxically is required for the generation and/or fixation of UV-induced frameshift mutations specifically in non-replicating cells.     

Adaptive Mutation in Saccharomyces cerevisiae

ABSTRACT

Adaptive mutation is a generic term for processes that allow individual cells of nonproliferating cell populations to acquire advantageous mutations and thereby to overcome the strong selective pressure of proliferation-limiting environmental conditions. Prerequisites for an occurrence of adaptive mutation are that the selective conditions are nonlethal and that a restart of proliferation may be accomplished by some genetic change in principle. The importance of adaptive mutation is derived from the assumption that it may, on the one hand, result in an accelerated evolution of microorganisms and, on the other, in multicellular organisms may contribute to a breakout of somatic cells from negative growth regulation, i.e., to cancerogenesis. Most information on adaptive mutation in eukaryotes has been gained with the budding yeast Saccharomyces cerevisiae. This review focuses comprehensively on adaptive mutation in this organism and summarizes our current understanding of this issue.     

环境胁迫诱导的细胞适应性突变

细胞具有普遍的突变和进化能力, 如病原菌的抗药性、工业菌株的适应性和人体细胞的癌变等, 但是细胞的适应性突变是如何产生的呢？通过非致死性突变分析模型的建立与应用, 产生了新的适应性进化观点, 即环境胁迫诱导细胞适应性突变。这种环境诱导的细胞突变过程涉及多方面的生理调控, 包括细胞内毒性物质(如氧活性物质)积累并造成DNA损伤、DNA错配修复的活性受到抑制、胞内RpoS反应和SOS反应被激活等。这些反应使胞内高保真的DNA复制状态转变为低保真的DNA修复状态, 提高胞内突变率和重组活性。此外, 基因转录影响基因组的不稳定, 容易产生DNA损伤, 并造成局部的高突变率, 即形成了转录偶联的DNA修复与突变为基础的适应性突变观点。文章围绕环境胁迫诱导细胞突变率增加和转录偶联的DNA修复与突变这两种适应性突变分子机制, 阐述其相关的研究进展, 以期更好地理解环境条件诱导细胞发生适应性突变的过程。     

Effect of growth under selection on appearance of chromosomal mutations in Salmonella enterica

Populations adapt physiologically using regulatory mechanisms and genetically by means of mutations that improve growth. During growth under selection, genetic adaptation can be rapid. In several genetic systems, the speed of adaptation has been attributed to cellular mechanisms that increase mutation rates in response to growth limitation. An alternative possibility is that growth limitation serves only as a selective agent but acts on small-effect mutations that are common under all growth conditions. The genetic systems that initially suggested stress-induced mutagenesis have been analyzed without regard for multistep adaptation and some include features that make such analysis difficult. To test the selection-only model, a simpler system is examined, whose behavior was originally attributed to stress-induced mutagenesis (Yang et al. 2001, 2006). A population with a silent chromosomal lac operon gives rise to Lac+ revertant colonies that accumulate over 6 days under selection. Each colony contains a mixture of singly and doubly mutant cells. Evidence is provided that the colonies are initiated by pre-existing single mutants with a weak Lac+ phenotype. Under selection, these cells initiate slow-growing clones, in which a second mutation arises and improves growth of the resulting double mutant. The system shows no evidence of general mutagenesis during selection. Selection alone may explain rapid adaptation in this and other systems that give the appearance of mutagenesis.     

Mismatch repair modulation of MutY activity drives Bacillus subtilis stationary-phase mutagenesis

Stress-promoted mutations that occur in nondividing cells (adaptive mutations) have been implicated strongly in causing genetic variability as well as in species survival and evolutionary processes. Oxidative stress-induced DNA damage has been associated with generation of adaptive His(+) and Met(+) but not Leu(+) revertants in strain Bacillus subtilis YB955 (hisC952 metB5 leuC427). Here we report that an interplay between MutY and MutSL (mismatch repair system [MMR]) plays a pivotal role in the production of adaptive Leu(+) revertants. Essentially, the genetic disruption of MutY dramatically reduced the reversion frequency to the leu allele in this model system. Moreover, the increased rate of adaptive Leu(+) revertants produced by a MutSL knockout strain was significantly diminished following mutY disruption. Interestingly, although the expression of mutY took place during growth and stationary phase and was not under the control of RecA, PerR, or σ(B), a null mutation in the mutSL operon increased the expression of mutY several times. Thus, in starved cells, saturation of the MMR system may induce the expression of mutY, disturbing the balance between MutY and MMR proteins and aiding in the production of types of mutations detected by reversion to leucine prototrophy. In conclusion, our results support the idea that MMR regulation of the mutagenic/antimutagenic properties of MutY promotes stationary-phase mutagenesis in B. subtilis cells.     

The effect of Spo0A and AbrB proteins on expression of the genes of guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis recombinant strains

Guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus are actively secreted under phosphate starvation by recombinant strains of Bacillus subtilis with native regulatory systems and by strains defective in some proteins of the Spo0A phosphorylation pathway. The level of expression of ribonuclease genes has been shown to increase approximately sixfold in recombinant strains with mutation in the spo0A gene and threefold in the spo0A/abrB mutants, as compared with native strains. These results demonstrate that the Spo0A protein regulates the production of ribonucleases and thus acts as a repressor, while the AbrB protein is an activator of expression of the genes encoding ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells.     

Single-strand-specific exonucleases prevent frameshift mutagenesis by suppressing SOS induction and the action of DinB/DNA polymerase IV in growing cells

Escherichia coli strains carrying null alleles of genes encoding single-strand-specific exonucleases ExoI and ExoVII display elevated frameshift mutation rates but not base substitution mutation rates. We characterized increased spontaneous frameshift mutation in ExoI- ExoVII- cells and report that some of this effect requires RecA, an inducible SOS DNA damage response, and the low-fidelity, SOS-induced DNA polymerase DinB/PolIV, which makes frameshift mutations preferentially. We also find that SOS is induced in ExoI- ExoVII- cells. The data imply a role for the single-stranded exonucleases in guarding the genome against mutagenesis by removing excess single-stranded DNA that, if left, leads to SOS induction and PolIV-dependent mutagenesis. Previous results implicated PolIV in E. coli mutagenesis specifically during starvation or antibiotic stresses. Our data imply that PolIV can also promote mutation in growing cells under genome stress due to excess single-stranded DNA.     

Oxidative DNA damage defense systems in avoidance of stationary-phase mutagenesis in Pseudomonas putida

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria.     

Genome-wide hypermutation in a subpopulation of stationary-phase cells underlies recombination-dependent adaptive mutation.

Stationary-phase mutation in microbes can produce selected (''adaptive'') mutants preferentially. In one system, this occurs via a distinct, recombination-dependent mechanism. Two points of controversy have surrounded these adaptive reversions of an Escherichia coli lac mutation. First, are the mutations directed preferentially to the selected gene in a Lamarckian manner? Second, is the adaptive mutation mechanism specific to the F plasmid replicon carrying lac? We report that lac adaptive mutations are associated with hypermutation in unselected genes, in all replicons in the cell. The associated mutations have a similar sequence spectrum to the adaptive reversions. Thus, the adaptive mutagenesis mechanism is not directed to the lac genes, in a Lamarckian manner, nor to the F'' replicon carrying lac. Hypermutation was not found in non-revertants exposed to selection. Therefore, the genome-wide hypermutation underlying adaptive mutation occurs in a differentiated subpopulation. The existence of mutable subpopulations in non-growing cells is important in bacterial evolution and could be relevant to the somatic mutations that give rise to cancers in multicellular organisms.     

Activation of the bgl operon by adaptive mutation

In growing Escherichia coli K12 cells, the cryptic bgl operon is activated 98% of the time by insertions of IS1 or IS5 into the control region, designated bglR. The activated bgl operon permits utilization of the beta-glucoside sugar arbutin as a sole carbon and energy source. The bgl operon is also activated by late-occurring mutations during prolonged selection on arbutin. The late-occurring mutations that occurred during prolonged carbon starvation in the presence of arbutin were "adaptive mutations" because they were specific to the presence of arbutin, and they did not occur during prolonged starvation in the absence of arbutin. The spectrum of late-arising mutations differed from that of early-arising, growth-dependent mutations in that 20% of the late-arising mutants resulted from mutations at the hns locus. This provides the first direct evidence for adaptive mutagenesis mediated by the insertion of IS elements. Because no special genetic background is required to select Bgl+ mutants, this affords the opportunity to study IS-element-mediated adaptive mutagenesis in a variety of genetic backgrounds, including the backgrounds of natural isolates of E. coli.      

Strand specificity for UV-induced DNA repair and mutations in the Chinese hamster HPRT gene.

DNA excision repair modulates the mutagenic effect of many genotoxic agents. The recently observed strand specificity for removal of UV-induced cyclobutane dimers from actively transcribed genes in mammalian cells could influence the nature and distribution of mutations in a particular gene. To investigate this, we have analyzed UV-induced DNA repair and mutagenesis in the same gene, i.e. the hypoxanthine phosphoribosyl-transferase (hprt) gene. In 23 hprt mutants from V79 Chinese hamster cells induced by 2 J/m2 UV we found a strong strand bias for mutation induction: assuming that pre-mutagenic lesions occur at dipyrimidine sequences, 85% of the mutations could be attributed to lesions in the nontranscribed strand. Analysis of DNA repair in the hprt gene revealed that more than 90% of the cyclobutane dimers were removed from the transcribed strand within 8 hours after irradiation with 10 J/m2 UV, whereas virtually no dimer removal could be detected from the nontranscribed strand even up to 24 hr after UV. These data present the first proof that strand specific repair of DNA lesions in an expressed mammalian gene is associated with a strand specificity for mutation induction.