Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or without general hypermutability

In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac(+) revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification-induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model.     

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.     

The yeast HSM3 gene acts in one of the mismatch repair pathways.

Mutants with enhanced spontaneous mutability (hsm) to canavanine resistance were induced by N-methyl-N-nitrosourea in Saccharomyces cerevisiae. One bearing the hsm3-1 mutation was used for this study. This mutation does not increase sensitivity to the lethal action of different mutagens. The hsm3-1 mutation produces a mutator phenotype, enhancing the rates of spontaneous mutation to canavanine resistance and reversions of lys1-1 and his1-7. This mutation increases the rate of intragenic mitotic recombination at the ADE2 gene. The ability of the hsm3 mutant to correct DNA heteroduplex is reduced in comparison with the wild-type strain. All these phenotypes are similar to ones caused by pms1, mlhl and msh2 mutations. In contrast to these mutations, hsm3-1 increases the frequency of ade mutations induced by 6-HAP and UV light. Epistasis analysis of double mutants shows that the PMS1 and HSM3 genes control different mismatch repair systems. The HSM3 gene maps to the right arm of chromosome II, 25 cM distal to the HIS7 gene. Strains that bear a deleted open reading frame YBR272c have the genetic properties of the hsm3 mutant. The HSM3 product shows weak similarity to predicted products of the yeast MSH genes (homologs of the Escherichia coli mutS gene). The HSM3 gene may be a member of the yeast MutS homolog family, but its function in DNA metabolism differs from the functions of other yeast MutS homologs.     

Interaction of the HSM3 gene with genes initiating homologous recombination repair in yeast Saccharomyces cerevisiae

It was assumed previously that the mutator phenotype of the hms3 mutant was determined by processes taking place in the D-loop. As a next step, genetic analysis was performed to study the interactions between the hsm3 mutation and mutations of the genes that control the initial steps of the D-loop formation. The mutations of the MMS4 and XRS2 genes, which initiate the double-strand break formation and subsequent repair, were shown to completely block HSM3-dependent UV-induced mutagenesis. Mutations of the RAD51, RAD52, and RAD54 genes, which are also involved in the D-loop formation, only slightly decreased the level of UV-induced mutagenesis in the hsm3 mutant. Similar results were observed for the interaction of hsm3 with the mph1 mutation, which stabilizes the D-loop. In contrast, the shu1 mutation, which destabilizes the D-loop structure, led to an extremely high level of UV-induced mutagenesis and displayed epistatic interactions with the hsm3 mutation. The results made it possible to assume that the hsm3 mutation destabilizes the D-loop, which is a key substrate of both Rad5- and Rad52-dependent postreplicative repair pathways.     

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.     

HSM2 (HMO1) gene participates in mutagenesis control in yeast Saccharomyces cerevisiae

We have previously reported about a new Saccharomyces cerevisiae mutation, hsm2-1, that results in increase of both spontaneous and UV-induced mutation frequencies but does not alter UV-sensitivity. Now HSM2 gene has been genetically and physically mapped and identified as a gene previously characterized as HMO1, a yeast homologue of human high mobility group genes HMG1/2. We found that hsm2 mutant is slightly deficient in plasmid-borne mismatch repair. We tested UV-induced mutagenesis in double mutants carrying hsm2-1 mutation and a mutation in a gene of principal damaged DNA repair pathways (rad2 and rev3) or in a mismatch repair gene (pms1 and recently characterized in our laboratory hsm3). The frequency of UV-induced mutations in hsm2 rev3 was not altered in comparison with single rev3 mutant. In contrast, the interaction of hsm2-1 with rad2 and pms1 was characterized by an increased frequency of UV-induced mutations in comparison with single rad2 and pms1 mutants. The UV-induced mutation frequency in double hsm2 hsm3 mutant was lower than in the single hsm2 and hsm3 mutants. The role of the HSM2 gene product in control of mutagenesis is discussed.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

High mutation rate of a long microsatellite allele in Drosophila melanogaster provides evidence for allele-specific mutation rates

Within recent years, microsatellite have become one of the most powerful genetic markers in biology. For several mammalian species, microsatellite mutation rates have been estimated on the order of 10(- 3)-10(-5). A recent study, however, demonstrated mutation rates in Drosophila melanogaster of at least one order of magnitude lower than those in mammals. To further test this result, we examined mutation rates of different microsatellite loci using a larger sample size. We screened 24 microsatellite loci in 119 D. melanogaster lines maintained for approximately 250 generations and detected 9 microsatellite mutations. The average mutation rate of 6.3 x 10(-6) is identical to the mutation rate from a previous study. Most interestingly, all nine mutations occurred at the same allele of one locus (DROYANETSB). This hypermutable allele has 28 dinucleotide repeats and is among the longest microsatellite reported in D. melanogaster. The allele-specific mutation rate of 3.0 x 10(-4) per generation is within the range of mammalian mutation rates. Future microsatellite analyses will have to account for the dramatic differences in allele-specific mutation rates.      

Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra

It is often assumed mutant frequencies, as measured in a DNA sample, faithfully represent basic mutation rates associated with these mutations. This paradigm was extremely helpful for in vitro studies of the mechanisms of mutagenesis/repair and causes of mutations. However, in vivo, mutant fractions appear to vary dramatically and randomly from sample to sample. It's unlikely that basic mutational rates vary so much. Such variations are probably caused by clonal expansions of mutants within tissue. Whether a particular tissue sample includes an expansion or not, is a matter of chance, which explains the observed random fluctuations of mutant fractions. Well-known examples of clonal expansions involve pathological conditions such as cancer or mitochondrial disease. It is less appreciated that even in normal tissue, expansions of somatic mutants create local deviations from the "expected" mutant frequencies. The sizes of clonal expansions appear to span a wide range and thus, may affect samples of various sizes, from individual cells to individuals. In conclusion, human body appears to be a sort of a "gambling ground" for clonally expanding mutants. We speculate that expansion of early mutants rather than de novo mutation at old age may be the major source of at least some aging-specific mutants in our bodies.     

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.     

A signature element distinguishes sibling and independent mutations in a shuttle vector plasmid.

We have developed a new shuttle vector plasmid for studying mutagenesis in mammalian cells that permits proof of independence of identical mutations. Mutations occur more frequently at some sites in a gene than in others, and in a collection of mutant plasmids from a single transfection of mammalian cells the same mutation may appear several times. However, those arising from independent events cannot be distinguished from siblings of an initial event. The new vector system (pSP189) is a population of plasmids, each of which contains an 8-bp 'signature sequence'. This sequence confers a unique identification tag to each plasmid and allows individual members to be identified by a distinctive signature. The plasmid also carries the Escherichia coli bacterial supF gene as a marker for mutagenesis, as well as sequences which support replication in primate (including human) cells and E. coli. We have used the pSP189 system to generate a UV-induced spectrum of mutations in supF following replication in a single plate of human DNA-repair-deficient cells (xeroderma pigmentosum, complementation group A). With the signature sequence, we were able to determine whether identical mutations derived from the transfection were of independent or sibling origin. There were eight identical mutations at the strongest hotspot, all of which had different signature sequences. Only one of these events would have been reported in previous experiments. This plasmid reduces the effort required to generate a spectrum of mutations caused by a DNA-damaging agent and allows a more accurate assessment of mutational hotspot intensity.     

Second-site suppression of RNase E essentiality by mutation of the deaD RNA helicase in Escherichia coli

Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.     

A mutation in the DNA adenine methylase gene (dam) of Salmonella typhimurium decreases susceptibility to 9-aminoacridine-induced frameshift mutagenesis

A mutant of Salmonella typhimurium with a reduced response to mutation induction by 9-aminoacridine (9AA) has been isolated. The mutation (dam-2) is located in the DNA adenine methylase gene. The dam-2 mutant strain exhibits a level of sensitivity to 2-aminopurine (2AP) intermediate between that of the dam+ and the DNA adenine methylation-deficit dam-1 strain, and 2AP sensitivity was reversed by introduction of a mutH mutation or of the plasmid pMQ148 (which carries a functional Escherichia coli dam+ gene). However, the dam-2 strain is not grossly defective in DNA adenine methylase activity. Whole cell DNA appears full methylated at -GATC- sites. The levels of 9AA required to induce equivalent levels of frameshift mutagenesis in the dam-2 strain were approximately 2-fold higher than for the dam+ strain. Introduction of pMQ148 dam+ reduced the level of 9AA required for induction of frameshift mutations 4-fold in the dam-2 strain and 2-fold in the dam+ strain. The dam-2 mutation had no effect on the levels of ICR191 required for induction of frameshift mutations, but introduction of pMQ148 reduced the ICR191-induced mutagenesis 2-fold. The dam+/pMQ148, dam-2/pMQ148 and dam-1/pMQ148 strains showed identical dose-response curves for both 9AA and ICR191. These results are consistent with a slightly reduced (dam-2) or increased (pMQ148) rate of methylation at the replication fork. The 2AP sensitivity of the dam-2 strain cannot be simply explained. Furthermore, addition of methionine to the assay medium reverses the 2AP sensitivity of the dam-2 strain, but has no effect on 9AA mutagenesis.     

适应性突变的遗传学特征

基于大肠杆菌FC40菌株的研究结果表明,适应性突变依赖RecBCD重组途径的酶,要求SOS反应的部分基因功能,lac+回复突变序列都是在单核苷酸短重复序列处的一个碱基缺失。有证据表明有的适应性突变来自一个或多个暂时性的超突变的细胞亚群,它们的基因组发生大量的突变,转座子高频丢失。产生这种暂时性的超突变的增变子可能是因为细胞的MMR活性暂时不足,或是因错误翻译产生丧失了校读活性的DNA聚合酶III。其他一些研究系统虽然得到了一些同FC40菌株不一致的结论,但所有实验证据都表明,在饥饿等环境胁迫因子作用下,非生长或缓慢生长的细胞可以产生突变,这种突变具有生长依赖的自发突变所不同的一些遗传学特征。 Abstract:The research based on the Escherichia coli FC40 showed that adaptive mutations required the enzymes of RecBCD recombination pathway and some unknown proteins of SOS response,and the mutation spectrum of lac+ revertants is single-base deletions in the small mononucleotide repeats.Some evidence showed that the revertants with adaptive mutations partly come from one (or some) subset of transient hypermutable subpopulation of cells,in which high frequently losing of transposons and genome-wide mutations were observed.It was suggested that this kind of transient hypermutability may be due to the transient deficient activity of mismatch repair (MMR) system,or a defective epsilon unit of DNA polymerase III generated by mistranslation.Although other systems demonstrated some different mechanisms from FC40,all research works suggested that,adaptive mutations occurred in nondividing or nongrowing cells under environmental stresses,for example,starvation,displayed different genetic features from growth-dependent spontaneous mutation.     

The amplification model for adaptive mutation: simulations and analysis

It has been proposed that the lac revertants arising under selective conditions in the Cairns experiment do not arise by stress-induced mutagenesis of stationary phase cells as has been previously assumed. Instead, these revertants may arise within growing clones initiated by cells with a preexisting duplication of the weakly functional lac allele used in this experiment. It is proposed that spontaneous stepwise increases in lac copy number (amplification) allow a progressive improvement in growth. Reversion is made more likely primarily by the resultant increase in the number of mutational targets--more cells with more lac copies. The gene amplification model requires no stress-induced variation in the rate or target specificity of mutation and thus does not violate neo-Darwinian theory. However, it does require that a multistep process of amplification, reversion, and amplification segregation be completed within approximately 20 generations of growth. This work examines the proposed amplification model from a theoretical point of view, formalizing it into a mathematical framework and using this to determine what would be required for the process to occur within the specified period. The analysis assumes no stress-induced change in mutation rate and describes only the growth improvement occurring during the process of amplification and subsequent elimination of excess mutant lac copies. The dynamics of the system are described using Monte Carlo simulations and numerical integration of the deterministic equations governing the system. The results imply that the amplification model can account for the behavior of the system using biologically reasonable parameter values and thus can, in principle, explain Cairnsian adaptive mutation.     

Characterization of cycA mutants of Escherichia coli. An assay for measuring in vivo mutation rates

Quantitative assessment of the spontaneous or induced genomic mutation rate, a fundamental evolutionary parameter, usually requires the use of well-characterized mutant selection systems. Although there is a great number of genetic selection schemes available in Escherichia coli, the selection of D-cycloserine resistant mutants is shown here to be particularly useful to yield a general view of mutation rates and spectra. The combination of a well-defined experimental protocol with the Ma-Sandri-Sarkar maximum likelihood method of fluctuation analysis results in reproducible data, adequate for statistical comparisons. The straightforward procedure is based on a simple phenotype-genotype relationship, and detects mutations in the single-copy, chromosomal cycA gene, involved in the uptake of D-cycloserine. In contrast to the widely used rifampicin resistance assay, the procedure selects mutations which are neutral in respect of cell growth. No specific genetic background is needed, and practically the entire mutation spectrum (base substitutions, frameshifts, deletions, insertions) can simultaneously be measured. A systematic analysis of cycA mutations revealed a spontaneous mutation rate of 6.54 x 10(-8) in E. coli K-12 MG1655. The mutation spectrum was dominated by point mutations (base substitutions, frameshifts), spread over the entire gene. IS insertions, caused by IS1, IS2, IS3, IS4, IS5 and IS150, represented 24% of the mutations.     

Suppressors of the secY24 mutation: identification and characterization of additional ssy genes in Escherichia coli

We previously reported (Shiba et al., J. Bacteriol. 160:696-701, 1984) the isolation and characterization of the mutation (ssy) that suppresses the protein export defect due to the secY24(Ts) mutation and causes cold-sensitive growth of Escherichia coli. This report describes more systematic isolation of ssy mutations. Among temperature-resistant revertants of the secY24 mutant, 65 mutants were found to be cold sensitive. These cold-sensitive mutations have been classified by genetic mapping. Twenty-two mutations fell into the ssyA class previously described. The remaining mutations were located at five new loci: ssyB at 9.5 min between tsx and lon; ssyD around 3 min; ssyE at 72.5 min near secY; ssyF at 20.5 min within rpsA; and ssyG at 69.0 min near argG. Two predominant classes, ssyA and ssyB, are probably affected in protein synthesis at the elongation step, whereas the ssyF mutant contained an altered form of ribosomal protein S1 (the gene product of rpsA). These cold-sensitive ssy mutations which suppress secY24 may define genes whose function is somehow involved in the secY-dependent protein secretion mechanism. However, the existence of multiple suppressor loci makes it unlikely that all of these genes specify additional components of the export machinery. A delicate balance may exist between the systems for synthesizing and exporting proteins.     

Isolation and mapping of a mutation in Escherichia coli with altered levels of ribonuclease H.

A mutant of Escherichia coli with altered levels of ribonuclease (RNase) H was isolated after mutagenesis with ethyl methane sulfonate. A procedure for assaying RNase H in partially purified extracts was used to screen approximately 1,500 colonies for variations in RNase H activity. Confirmation of a lower level of RNase H in the mutant was accomplished by analysis of RNase H in sodium dodecyl sulfate-polyacrylamide gels. By Hfr, F', and P1 transduction mapping, the genetic locus responsible for the lower levels of RNase H was located at 5.1 min on the E. coli chromosome. This mutation (rnh) represents a new locus on the E. coli chromosome. The only phenotypic characteristic of this mutation which has been observed to date is the lower level of RNase H (30% of parental values).