Abstract: Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.
Abstract: Maintenance of genomic stability is critical for all cells. Homologous recombination (HR) pathways promote genome stability using evolutionarily conserved proteins such as RecA, SSB, and RecQ, the Escherichia coli homologue of five human proteins at least three of which suppress genome instability and cancer. A previous report indicated that RecQ promotes the net accumulation in cells of intermolecular HR intermediates (IRIs), a net effect opposite that of the yeast and two human RecQ homologues. Here we extend those conclusions. We demonstrate that cells that lack both UvrD, an inhibitor of RecA-mediated strand exchange, and RecG, a DNA helicase implicated in IRI resolution, are inviable. We show that the uvrD recG cells die a "death-by-recombination" in which IRIs accumulate blocking chromosome segregation. First, their death requires RecA HR protein. Second, the death is accompanied by cytogenetically visible failure to segregate chromosomes. Third, FISH analyses show that the unsegregated chromosomes have completed replication, supporting the hypothesis that unresolved IRIs prevented the segregation. Fourth, we show that RecQ and induction of the SOS response are required for the accumulation of replicated, unsegregated chromosomes and death, as are RecF, RecO, and RecJ. ExoI exonuclease and MutL mismatch-repair protein are partially required. This set of genes is similar but not identical to those that promote death-by-recombination of DeltauvrD Deltaruv cells. The data support models in which RecQ promotes the net accumulation in cells of IRIs and RecG promotes resolution of IRIs that form via pathways not wholly identical to those that produce the IRIs resolved by RuvABC. This implies that RecG resolves intermediates other than or in addition to standard Holliday junctions resolved by RuvABC. The role of RecQ in net accumulation of IRIs may be shared by one or more of its human homologues.
Abstract: Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA-damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS-inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans.
Abstract: Summary Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by 'point' mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, sigma(E) acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of sigma(32), which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that sigma(E) promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by sigma(E), and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.
Abstract: Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(o(c)) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(o(c)) alleles fully suppress the phenotype of constitutively SOS-"off" lexA(Ind(-)) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(o(c)) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition.
Abstract: Slade et al. (2009) describe in this issue how the genome of the bacterium Deinococcus radiodurans gets reassembled after being shattered by high-dose radiation. In contrast to the extreme nature of the damage, the steps of repair appear surprisingly ordinary. So, why can't all organisms carry out extreme genome repair?
Abstract: Deletions and duplications of chromosomal segments (copy number variants, CNVs) are a major source of variation between individual humans and are an underlying factor in human evolution and in many diseases, including mental illness, developmental disorders and cancer. CNVs form at a faster rate than other types of mutation, and seem to do so by similar mechanisms in bacteria, yeast and humans. Here we review current models of the mechanisms that cause copy number variation. Non-homologous end-joining mechanisms are well known, but recent models focus on perturbation of DNA replication and replication of non-contiguous DNA segments. For example, cellular stress might induce repair of broken replication forks to switch from high-fidelity homologous recombination to non-homologous repair, thus promoting copy number change.
Abstract: In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations are a common route to clinically relevant beta-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial beta-lactam resistance mutations using the well-developed genetics of E. coli by integrating enterobacterial ampRC genes into the E. coli chromosome. Like the cells of other enterobacteria, E. coli cells acquire beta-lactam resistance by ampD mutation. Here we show that starvation and stress responses provoke ampD beta-lactam resistance mutagenesis. When starved on lactose medium, Lac(-) strains used in mutagenesis studies accumulate ampD beta-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this mutagenesis, in agreement with the results obtained for lac reversion in these cells. Surprisingly, the stress-induced ampD mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike lac mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as starvation, can induce clinically relevant resistance mutations. Finally, we used the ampD system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism.
Abstract: The Escherichia coli chromosome encodes seven demonstrated type 2 toxin-antitoxin (TA) systems: cassettes of two or three cotranscribed genes, one encoding a stable toxin protein that can cause cell stasis or death, another encoding a labile antitoxin protein, and sometimes a third regulatory protein. We demonstrate that the yafNO genes constitute an additional chromosomal type 2 TA system that is upregulated during the SOS DNA damage response. The yafNOP genes are part of the dinB operon, of which dinB underlies stress-induced mutagenesis mechanisms. yafN was identified as a putative antitoxin by homology to known antitoxins, implicating yafO (and/or yafP) as a putative toxin. Using phage-mediated cotransduction assays for linkage disruption, we show first that yafN is an essential gene and second that it is essential only when yafO is present. Third, yafP is not a necessary part of either the toxin or the antitoxin. Fourth, although DinB is required, the yafNOP genes are not required for stress-induced mutagenesis in the Escherichia coli Lac assay. These results imply that yafN encodes an antitoxin that protects cells against a yafO-encoded toxin and show a protein-based TA system upregulated by the SOS response.
Abstract: In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac(+) mutants, with and without evidence of descent from the HMS, have similar Lac(+) mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac(+) mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.
Abstract: The RecQ-helicase family is widespread, is highly conserved, and includes human orthologs that suppress genomic instability and cancer. In vivo, some RecQ homologs promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that, in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promotes this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologs.
Abstract: BACKGROUND: Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3'-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands. METHODOLOGY/PRINCIPLE FINDINGS: This paper re-examines heteroduplex-patch-strand polarity using phage lambda and the lambdadv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for lambda recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5' or 3' DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation. CONCLUSIONS/SIGNIFICANCE: These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism.
Abstract: Spontaneous DNA breakage is predicted to be a frequent, inevitable consequence of DNA replication and is thought to underlie much of the genomic change that fuels cancer and evolution. Despite its importance, there has been little direct measurement of the amounts, types, sources and fates of spontaneous DNA lesions in living cells. We present a direct, sensitive flow cytometric assay in single living Escherichia coli cells for DNA lesions capable of inducing the SOS DNA damage response, and we report its use in quantification of spontaneous DNA double-strand breaks (DSBs). We report efficient detection of single chromosomal DSBs and rates of spontaneous breakage approximately 20- to 100-fold lower than predicted. In addition, we implicate DNA replication in the origin of spontaneous DSBs with the finding of fewer spontaneous DSBs in a mutant with altered DNA polymerase III. The data imply that spontaneous DSBs induce genomic changes and instability 20-100 times more potently than previously appreciated. Finally, FACS demonstrated two main cell fates after spontaneous DNA damage: viability with or without resumption of proliferation.
Abstract: Our concept of a stable genome is evolving to one in which genomes are plastic and responsive to environmental changes. Growing evidence shows that a variety of environmental stresses induce genomic instability in bacteria, yeast, and human cancer cells, generating occasional fitter mutants and potentially accelerating adaptive evolution. The emerging molecular mechanisms of stress-induced mutagenesis vary but share telling common components that underscore two common themes. The first is the regulation of mutagenesis in time by cellular stress responses, which promote random mutations specifically when cells are poorly adapted to their environments, i.e., when they are stressed. A second theme is the possible restriction of random mutagenesis in genomic space, achieved via coupling of mutation-generating machinery to local events such as DNA-break repair or transcription. Such localization may minimize accumulation of deleterious mutations in the genomes of rare fitter mutants, and promote local concerted evolution. Although mutagenesis induced by stresses other than direct damage to DNA was previously controversial, evidence for the existence of various stress-induced mutagenesis programs is now overwhelming and widespread. Such mechanisms probably fuel evolution of microbial pathogenesis and antibiotic-resistance, and tumor progression and chemotherapy resistance, all of which occur under stress, driven by mutations. The emerging commonalities in stress-induced-mutation mechanisms provide hope for new therapeutic interventions for all of these processes.
Abstract: 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.
Abstract: Special mechanisms of mutation are induced during growth-limiting stress and can generate adaptive mutations that permit growth. These mechanisms may provide improved models for mutagenesis in antibiotic resistance, evolution of pathogens, cancer progression and chemotherapy resistance. Stress-induced reversion of an Escherichia coli episomal lac frameshift allele specifically requires DNA double-strand-break-repair (DSBR) proteins, the SOS DNA-damage response and its error-prone DNA polymerase, DinB. We distinguished two possible roles for the DSBR proteins. Each might act solely upstream of SOS, to create single-strand DNA that induces SOS. This could upregulate DinB and enhance mutation globally. Or any or all of them might function other than or in addition to SOS promotion, for example, directly in error-prone DSBR. We report that in cells with SOS genes derepressed constitutively, RecA, RuvA, RuvB, RuvC, RecF, and TraI remain required for stress-induced mutation, demonstrating that these proteins act other than via SOS induction. RecA and TraI also act by promoting SOS. These and additional results with hyper-mutating recD and recG mutants support roles for these proteins via error-prone DSBR. Such mechanisms could localize stress-induced mutagenesis to small genomic regions, a potentially important strategy for adaptive evolution, both for reducing additional deleterious mutations in rare adaptive mutants and for concerted evolution of genes.
Abstract: Gene amplification is a collection of processes whereby a DNA segment is reiterated to multiple copies per genome. It is important in carcinogenesis and resistance to chemotherapeutic agents, and can underlie adaptive evolution via increased expression of an amplified gene, evolution of new gene functions, and genome evolution. Though first described in the model organism Escherichia coli in the early 1960s, only scant information on the mechanism(s) of amplification in this system has been obtained, and many models for mechanism(s) were possible. More recently, some gene amplifications in E. coli were shown to be stress-inducible and to confer a selective advantage to cells under stress (adaptive amplifications), potentially accelerating evolution specifically when cells are poorly adapted to their environment. We focus on stress-induced amplification in E. coli and report several findings that indicate a novel molecular mechanism, and we suggest that most amplifications might be stress-induced, not spontaneous. First, as often hypothesized, but not shown previously, certain proteins used for DNA double-strand-break repair and homologous recombination are required for amplification. Second, in contrast with previous models in which homologous recombination between repeated sequences caused duplications that lead to amplification, the amplified DNAs are present in situ as tandem, direct repeats of 7-32 kilobases bordered by only 4 to 15 base pairs of G-rich homology, indicating an initial non-homologous recombination event. Sequences at the rearrangement junctions suggest nonhomologous recombination mechanisms that occur via template switching during DNA replication, but unlike previously described template switching events, these must occur over long distances. Third, we provide evidence that 3'-single-strand DNA ends are intermediates in the process, supporting a template-switching mechanism. Fourth, we provide evidence that lagging-strand templates are involved. Finally, we propose a novel, long-distance template-switching model for the mechanism of adaptive amplification that suggests how stress induces the amplifications. We outline its possible applicability to amplification in humans and other organisms and circumstances.
Abstract: 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.
Abstract: The physiological role of topoisomerase III is unclear for any organism. We show here that the removal of topoisomerase III in temperature sensitive topoisomerase IV mutants in Escherichia coli results in inviability at the permissive temperature. The removal of topoisomerase III has no effect on the accumulation of catenated intermediates of DNA replication, even when topoisomerase IV activity is removed. Either recQ or recA null mutations, but not helD null or lexA3, partially rescued the synthetic lethality of the double topoisomerase III/IV mutant, indicating a role for topoisomerase III in recombination. We find a bias against deleting the gene encoding topoisomerase III in ruvC53 or DeltaruvABC backgrounds compared with the isogenic wild-type strains. The topoisomerase III RuvC double mutants that can be constructed are five- to 10-fold more sensitive to UV irradiation and mitomycin C treatment and are twofold less efficient in transduction efficiency than ruvC53 mutants. The overexpression of ruvABC allows the construction of the topoisomerase III/IV double mutant. These data are consistent with a role for topoisomerase III in disentangling recombination intermediates as an alternative to RuvABC to maintain the stability of the genome.
Abstract: The neo-Darwinists suggested that evolution is constant and gradual, and thus that genetic changes that drive evolution should be too. However, more recent understanding of phenomena called adaptive mutation in microbes indicates that mutation rates can be elevated in response to stress, producing beneficial and other mutations. We review evidence that, in Escherichia coli, two separate mechanisms of stress-induced genetic change occur that revert a lac frameshift allele allowing growth on lactose medium. First, compensatory frameshift ("point") mutations occur by a mechanism that includes DNA double-strand breaks and (we have suggested) their error-prone repair. Point mutation requires induction of the RpoS-dependent general stress response, and the SOS DNA damage response leading to upregulation of the error-prone DNA polymerase DinB (Pol IV), and occurs during a transient limitation of post-replicative mismatch repair activity. A second mechanism, adaptive amplification, entails amplification of the leaky lac allele to 20-50 tandem repeats. These provide sufficient beta-galactosidase activity for growth, thereby apparently deflecting cells from the point mutation pathway. Unlike point mutation, amplification neither occurs in hypermutating cells nor requires SOS or DinB, but like point mutation, amplification requires the RpoS-dependent stress response. Similar processes are being found in other bacterial systems and yeast. Stress-induced genetic changes may underlie much of microbial evolution, pathogenesis and antibiotic resistance, and also cancer formation, progression and drug resistance.
Abstract: Microbial cells under growth-limiting stress can generate mutations by mechanisms distinct from those in rapidly growing cells. These mechanisms might be specific stress responses that increase mutation rates, potentially altering rates of evolution, or might reflect non-stress-specific processes in rare growing cells. In an Escherichia coli model system, both frameshift reversion mutations and gene amplifications occur as apparent starvation-induced mutations. Whereas frameshift reversion ("point mutation") requires recombination proteins, the SOS response, and error-prone DNA polymerase IV (DinB), amplification requires neither SOS nor pol IV. We report that both point mutation and amplification require the stationary-phase and general stress response transcription factor RpoS (sigmaS). Growth-dependent mutation does not. Alternative interpretations are excluded. The results imply, first, that point mutation and amplification are stress responses that occur in differentiated stationary-phase (not rare growing) cells and, second, that transient genetic instability, producing both point mutation and genome rearrangement, may be a previously unrecognized component of the RpoS-dependent general stress response.
Abstract: Many recombination, DNA repair and DNA replication mutants have high basal levels of SOS expression as determined by a sulAp-lacZ reporter gene system on a population of cells. Two opposing models to explain how the SOS expression is distributed in these cells are: (i) the 'Uniform Expression Model (UEM)' where expression is evenly distributed in all cells or (ii) the 'Two Population Model (TPM)' where some cells are highly induced while others are not at all. To distinguish between these two models, a method to quantify SOS expression in individual bacterial cells was developed by fusing an SOS promoter (sulAp) to the green fluorescent protein (gfp) reporter gene and inserting it at attlambda on the Escherichia coli chromosome. It is shown that the fluorescence in sulAp-gfp cells is regulated by RecA and LexA. This system was then used to distinguish between the two models for several mutants. The patterns displayed by priA, dnaT, recG, uvrD, dam, ftsK, rnhA, polA and xerC mutants were explained best by the TPM while only lexA (def), lexA3 (ind-) and recA defective mutants were explained best by the UEM. These results are discussed in a context of how the processes of DNA replication and recombination may affect cells in a population differentially.
Abstract: As do many temperate bacteriophages, integrating conjugative elements (ICEs) recruit the SOS DNA damage response to mobilize themselves from the bacterial chromosome and infect other cells. This transfers resistance to multiple antibiotics. Several commonly used antibiotics induce the SOS response, potentially hastening genetic change and the evolution to resistance of pathogenic populations. The use of such antibiotics should be reconsidered.
Abstract: "Adaptive mutation" denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase lac(-)Escherichia coli cells on lactose medium induces Lac(+)revertants at higher frequencies than predicted by usual mutation models. These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies (500-10,000 cells), we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac(+)cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v)lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.
Abstract: Apparently conflicting data regarding the role of SOS-inducible, error-prone DNA polymerase IV (DinB) in spontaneous mutation are resolved by the finding that mutation is reduced by a polar allele with which dinB and neighboring yafN are deleted but not by two nonpolar dinB alleles. We demonstrate the existence of a dinB operon that contains four genes, dinB-yafN-yafO-yafP. The results imply a role for yafN, yafO, and/or yafP in spontaneous mutation.
Abstract: Single-strand-dependent DNA exonucleases play important roles in DNA repair and recombination in all organisms. In Escherichia coli the redundant functions provided by the RecJ, ExoI, ExoVII and ExoX exonucleases are required for mismatch repair, UV resistance and homologous recombination. We have examined whether the xni gene product, the single-strand exonuclease ExoIX, is also a member of this group. We find that deletion of xni has no effect on the above processes, or on resistance to oxidative damage, even in combination with other exonuclease mutations. We conclude that the xni gene product does not belong to this group of nucleases that play redundant roles in DNA recombination and repair.
Abstract: In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations in the ampD gene are a common route to beta-lactam antibiotic resistance. We constructed an assay system for studying mechanism(s) of enterobacterial ampD mutation using the well-developed genetics of E. coli. We integrated the Enterobacter ampRC genes into the E. coli chromosome. These cells acquire spontaneous recombination- and SOS response-independent beta-lactam resistance mutations in ampD. This chromosomal system is useful for studying mutation mechanisms that promote antibiotic resistance.
Abstract: "Adaptive" or "stationary-phase" mutation is a collection of apparent stress responses in which cells exposed to a growth-limiting environment generate genetic changes, some of which can allow resumption of rapid growth. In the well-characterized Lac system of Escherichia coli, reversions of a lac frameshift allele give rise to adaptive point mutations. Also in this system, adaptive gene amplification has been documented as a separate and parallel response that allows growth on lactose medium without acquisition of a compensatory frameshift mutation. In amplification, the DNA region containing the weakly functional lac allele becomes amplified to multiple copies, which produce sufficient enzyme activity to allow growth on the otherwise growth-limiting lactose medium. The amplifications are "adaptive" in that they occur after cells encounter the growth-limiting environment. Adaptive amplification is a reversible genetic change that allows adaptation and growth. It may be similar to chromosomal instability observed in the origins and progression of many cancers. We explore possible molecular mechanisms of adaptive amplification in the bacterial system and note parallels to chromosomal instability in other systems.
Abstract: A basic principle of genetics is that the likelihood that a particular mutation occurs is independent of its phenotypic consequences. The concept of adaptive mutation seemed to challenge this principle with the discoveries of mutations stimulated by stress, some of which allow adaptation to the stress. The emerging mechanisms of adaptive genetic change cast evolution, development and heredity into a new perspective, indicating new models for the genetic changes that fuel these processes.
Abstract: Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a lac frameshift allele on an F' plasmid in Escherichia coli. The stationary-phase mutation mechanism at lac requires recombination proteins of the RecBCD double-strand-break repair system and the inducible error-prone DNA polymerase IV, and the mutations are mostly -1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F' lac. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.
Abstract: "Adaptive" or "stationary-phase" mutation is a collection of stress responses promoting mutations, some of which are advantageous. In 2000 and 2001, in Escherichia coli, adaptive gene amplification was documented, and a parallel adaptive point-mutation mechanism was linked to the error-prone DNA polymerase, DinB (pol IV). We suggest that DinB homologues may contribute to adaptive strategies of pathogens, including antigenic variation.
Abstract: Adaptive point mutation and amplification are induced responses to environmental stress, promoting genetic changes that can enhance survival. A specialized adaptive mutation mechanism has been documented in one Escherichia coli assay, but its enzymatic basis remained unclear. We report that the SOS-inducible, error-prone DNA polymerase (pol) IV, encoded by dinB, is required for adaptive point mutation in the E. coli lac operon. A nonpolar dinB mutation reduces adaptive mutation frequencies by 85% but does not affect adaptive amplification, growth-dependent mutation, or survival after oxidative or UV damage. We show that pol IV, together with the major replicase, pol III, can account for all adaptive point mutations at lac. The results identify a role for pol IV in inducible genetic change.
Abstract: Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P(BAD)), P(BAD) plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.
Abstract: Adaptive (or stationary-phase) mutation is a group of phenomena in which mutations appear to occur more often when selected than when not. They may represent cellular responses to the environment in which the genome is altered to allow survival. The best-characterized assay system and mechanism is reversion of a lac allele on an F' sex plasmid in Escherichia coli, in which the stationary-phase mutability requires homologous recombination functions. A key issue has concerned whether the recombination-dependent mutation mechanism is F' specific or is general. Hypermutation of chromosomal genes occurs in association with adaptive Lac(+) mutation. Here we present evidence that the chromosomal hypermutation is promoted by recombination. Hyperrecombinagenic recD cells show elevated chromosomal hypermutation. Further, recG mutation, which promotes accumulation of recombination intermediates proposed to prime replication and mutation, also stimulates chromosomal hypermutation. The coincident mutations at lac (on the F') and chromosomal genes behave as independent events, whereas coincident mutations at lac and other F-linked sites do not. This implies that transient covalent linkage of F' and chromosomal DNA (Hfr formation) does not underlie chromosomal mutation. The data suggest that recombinational stationary-phase mutation occurs in the bacterial chromosome and thus can be a general strategy for programmed genetic change.
Abstract: Upon starvation some Escherichia coli cells undergo a transient, genome-wide hypermutation (called adaptive mutation) that is recombination-dependent and appears to be a response to a stressful environment. Adaptive mutation may reflect an inducible mechanism that generates genetic variability in times of stress. Previously, however, the regulatory components and signal transduction pathways controlling adaptive mutation were unknown. Here we show that adaptive mutation is regulated by the SOS response, a complex, graded response to DNA damage that includes induction of gene products blocking cell division and promoting mutation, recombination, and DNA repair. We find that SOS-induced levels of proteins other than RecA are needed for adaptive mutation. We report a requirement of RecF for efficient adaptive mutation and provide evidence that the role of RecF in mutation is to allow SOS induction. We also report the discovery of an SOS-controlled inhibitor of adaptive mutation, PsiB. These results indicate that adaptive mutation is a tightly regulated response, controlled both positively and negatively by the SOS system.
Abstract: The radC102 mutation causes mild UV and X-ray sensitivity and was mapped previously to near pyrE and recG at 82 min on the Escherichia coli chromosome (I. Felzenszwalb, N. J. Sargentini, and K. C. Smith, Radiat. Res. 97:615-625, 1984). We report that radC102 has two striking phenotypes characteristic of recG mutations. First, it causes dramatically increased RecA-dependent mutation in a stationary-phase mutation assay. Second, it causes extreme UV sensitivity in combination with ruv mutations affecting the RuvABC Holliday junction resolution system. DNA sequencing of the radC and recG genes in radC102 strains revealed that the radC102 mutation creates a stop codon in recG that is predicted to truncate the RecG protein at 410 of 603 amino acids. A low-copy-number plasmid carrying the radC(+) gene did not affect the UV sensitivity of a wild-type strain, a radC102 strain, or a recG258::Tn10mini-kan strain. We conclude that radC102 is an allele of recG and that the function of the RadC protein remains to be determined.
Abstract: Adaptive mutation is an induced response to environmental stress in which mutation rates rise, producing permanent genetic changes that can adapt cells to stress. This contrasts with neo-Darwinian views of genetic change rates blind to environmental conditions. DNA amplification is a flexible, reversible genomic change that has long been postulated to be adaptive. We report the discovery of adaptive amplification at the lac operon in Escherichia coli. Additionally, we find that adaptive amplification is separate from, and does not lead to, adaptive point mutation. This contradicts a prevailing alternative hypothesis whereby adaptive mutation is normal mutability in amplified DNA. Instead, adaptive mutation and amplification are parallel routes of inducible genetic instability allowing rapid evolution under stress, and escape from growth inhibition.
Abstract: Stationary-phase mutation (a subset of which was previously called adaptive mutation) occurs in apparently nondividing, stationary-phase cells exposed to a nonlethal genetic selection. In one experimental system, stationary-phase reversion of an Escherichia coli F'-borne lac frameshift mutation occurs by a novel molecular mechanism that requires homologous recombination functions of the RecBCD system. Chromosomal mutations at multiple loci are detected more frequently in Lac+ stationary-phase revertants than in cells that were also exposed to selection but did not become Lac+. Thus, mutating cells represent a subpopulation that experiences hypermutation throughout the genome. This paper summarizes current knowledge regarding stationary-phase mutation in the lac system. Hypotheses for the mechanism of chromosomal hypermutation are discussed, and data are presented that exclude one hypothetical mechanism in which chromosomal mutations result from Hfr formation.
Abstract: DNA double-strand-break repair (DSBR) is, in many organisms, accomplished by homologous recombination. In Escherichia coli DSBR was thought to result from breakage and reunion of parental DNA molecules, assisted by known endonucleases, the Holliday junction resolvases. Under special circumstances, for example, SOS induction, recombination forks were proposed to initiate replication. We provide physical evidence that this is a major alternative mechanism in which replication copies information from one chromosome to another generating recombinant chromosomes in normal cells in vivo. This alternative mechanism can occur independently of known Holliday junction cleaving proteins, requires DNA polymerase III, and produces recombined DNA molecules that carry newly replicated DNA. The replicational mechanism underlies about half the recombination of linear DNA in E. coli; the other half occurs by breakage and reunion, which we show requires resolvases, and is replication-independent. The data also indicate that accumulation of recombination intermediates promotes replication dramatically.
Abstract: This paper is an invited Response to a recent Commentary [P.L. Foster, Rev. Mut. Res. 436 (1999) 179-184] entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking". The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation. A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation. In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time. Here we show further data that illustrate why this must be done to ensure accurate mutation measurements. Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation. We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity. Those data are substantial. The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there.
Abstract: In vitro, the methyl-directed mismatch repair system of Escherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.
Abstract: Major advances in understanding the molecular mechanism of recombination-dependent stationary-phase mutation in Escherichia coli occurred this past year. These advances are reviewed here, and we also present new evidence that the mutagenic state responsible is transient. We find that most stationary-phase mutants do not possess a heritable stationary-phase mutator phenotype, although a small proportion of heritable mutators was found previously. We outline similarities between this well-studied system and several recent examples of adaptive evolution associated with heritable mutator phenotype in a similarly small proportion of survivors of selection in nature and in the lab. We suggest the following: (1) Transient mutator states may also be a predominant source of adaptive mutations in these latter systems, the heritable mutators being a minority (Rosenberg 1997); (2) heritable mutators may sometimes be a product of, rather than the cause of, hypermutation that gives rise to adaptive mutations.
Abstract: 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.
Abstract: The sequences of adaptive reversions of a lac frameshift mutation in Escherichia coli resemble DNA polymerase errors, and the adaptive reversions decrease in strains with an antimutator DNA polymerase III (PolIII) allele. The latter finding could imply that DNA PolIII itself makes adaptive mutations. Alternatively, normal DNA PolIII errors could saturate post-synthesis mismatch repair during adaptive mutation. If so, the antimutator strain would produce fewer adaptive mutations because it possesses greater capacity for mismatch repair which could correct errors made by a polymerase other than DNA PolIII. Mismatch repair capacity is limited specifically during adaptive mutation, necessitating a test of this indirect model. This indirect model is ruled out here by the observation that the antimutator PolIII allele decreases adaptive mutation even in mismatch repair-defective cells. This supports a direct role for DNA PolIII in recombination-dependent adaptive mutation.
Abstract: Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase ("adaptive") mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.
Abstract: Adaptive mutations appear in response to selection. In the best-studied system, the two most controversial issues were resolved this year. The mutations are neither Lamarckian nor a peculiarity of bacterial sex, as had been suggested. They occur genome-wide in a hypermutable subpopulation of stressed cells. Genomic 'hot' and 'cold' regions may explain previous failures to detect similar mutations in other systems and at other sites. Stationary phase specific limitation of mismatch repair has also been discovered.
Abstract: This paper focuses on elucidation of the structures of intermediates in recombination stimulated by Chi recombination hotspots in vivo. We report that null mutations in genes encoding single-strand exonucleases of 3' polarity, Exonuclease I (Exo I), of 5' polarity, RecJ, and of both polarities, Exo VII, alter the ability of Chi sites to promote recombination, and diminish the frequency of recombination. Maximal effects occur when single-strand exonucleases of both polarities are eliminated. These data imply that 3' and 5' single-strand DNA ends, the substrates for these exonucleases, exist in bona fide, product-generating intermediates in Chi-stimulated recombination in vivo. These results also identify three new proteins not known previously to affect RecBCD-mediated recombination.
Abstract: In yeast meiotic recombination, alleles used as genetic markers fall into two classes as regards their fate when incorporated into heteroduplex DNA. Normal alleles are those that form heteroduplexes that are nearly always recognized and corrected by the mismatch repair system operating in meiosis. High PMS (postmeiotic segregation) alleles form heteroduplexes that are inefficiently mismatch repaired. We report that placing any of several high PMS alleles very close to normal alleles causes hyperrecombination between these markers. We propose that this hyperrecombination is caused by the high PMS allele blocking a mismatch repair tract initiated from the normal allele, thus preventing corepair of the two alleles, which would prevent formation of recombinants. The results of three point crosses involving two PMS alleles and a normal allele suggest that high PMS alleles placed between two alleles that are normally corepaired block that corepair.
Abstract: Over the past 6 years an unexpected way of making mutations in bacteria has challenged concepts of the genetic mechanisms behind evolution. Mechanistic studies of these so called 'adaptive' mutations are revealing a novel molecular mechanism involving DNA double-strand breaks, genetic recombination, probable DNA polymerase errors, and the possible suspension of mismatch repair during the reversion of a lac frameshift mutation in Escherichia coli. The molecular details of this process are altering our understanding of how mutations form in non-dividing cells.
Abstract: Aspects of the molecular mechanism of "adaptive" mutation are emerging from one experimental system: reversion of an Escherichia coli lac frameshift mutation carried on a conjugative plasmid. Homologous recombination is required and the mutations resemble polymerase errors. Reports implicating a role for conjugal transfer proteins suggested that the mutation mechanism is ordinary replication error occurring during transfer synthesis, followed by conjugation-like recombination, to capture the replicated fragment into an intact replicon. Whereas conjugational recombination uses either of two systems of Holliday junction resolution, we find that the adaptive lac reversions are inhibited by one resolution system and promoted by the other. Moreover, temporary absence of both resolution systems promotes mutation. These results imply that recombination intermediates themselves promote the mutations.
Abstract: In one experimental system, several handles on the molecular mechanism of apparent adaptive mutation have emerged. The system is reversion of a lac frameshift mutation in Escherichia coli. The molecular handles include a requirement for homologous recombination; the implication of DNA double-strand breaks as a molecular intermediate; a unique sequence spectrum of -1 deletions in mononucleotide repeats which implies polymerase errors, and also implies a failure of postsynthesis mismatch repair on those errors; and the involvement of sexual functions at some stage of the process. These molecular handles are revealing an unexpected new mechanism of mutagenesis.
Abstract: Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous. The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E. coli occurs in the absence of mismatch repair. The postulate that mismatch repair is disabled transiently during adaptive mutation in E. coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth. Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution.
Abstract: The genetic requirements for adaptive mutation in Escherichia coli parallel those for homologous recombination in the RecBCD pathway. Recombination-deficient recA and recB null mutant strains are deficient in adaptive reversion. A hyper-recombinagenic recD strain is hypermutable, and its hypermutation depends on functional recA and recB genes. Genes of subsidiary recombination systems are not required. These results indicate that the molecular mechanism by which adaptive mutation occurs includes recombination. No such association is seen for spontaneous mutation in growing cells.
Abstract: Adaptive reversion of a +1 frameshift mutation in Escherichia coli, which requires homologous recombination functions, is shown here to occur by -1 deletions in regions of small mononucleotide repeats. This pattern makes improbable recombinational mechanisms for adaptive mutation in which blocks of sequences are transferred into the mutating gene, and it supports mechanisms that use DNA polymerase errors. The pattern appears similar to that of mutations found in yeast cells and in hereditary colon cancer cells that are deficient in mismatch repair. These results suggest a recombinational mechanism for adaptive mutation that functions through polymerase errors that persist as a result of a deficiency in post-synthesis mismatch repair.
Abstract: An unexpected way of making mutations in bacteria has challenged concepts of the genetic mechanisms behind evolution for the past 6 years. Work on the molecular mechanism of these so called "adaptive" mutations is rapidly revealing a surprising and novel molecular mechanism, and it is altering our understanding of how mutations form in nondividing cells.
Abstract: In recent years two different styles of model for homologous recombination have been discussed, depending on whether or not the recombination event occurs in the vicinity of a double-strand break in DNA. The models of Holliday and Meselson and Radding exemplify those that do not involve a break whereas the model of Szostak et al is taken as an example of those that do. Recent advances in understanding a prototypic recombination system thought to promote exchange distant from DNA ends, at Chi sites, suggest a mechanism of initiation neither like Holliday/Meselson-Radding nor like Szostak et al. In those models, only one strand of DNA may invade a homologous DNA molecule. We propose a model for Chi in which exonuclease degrades DNA from a double-strand break to the Chi site; the exonuclease is converted into a helicase upon interaction with Chi; unwinding produces a recombinagenic split-end, and both 3'- and 5'-ending strands at the split-end are capable of invading a homologue. Different genetic consequences are proposed to result from invasion by each. We review evidence supporting the split-end model and suggest its application in at least some cases previously considered to proceed via the Meselson/Radding model and by the double-strand-break repair model of Szostak et al.
Abstract: Heteroduplex patch recombinants have received information in one DNA chain but have not recombined flanking markers. Evidence regarding which chain is exchanged bears on the structure of recombination intermediates. The direction of travel along DNA of RecBCD recombinase, the central enzyme in the Escherichia coli RecBCD pathway of homologous recombination, is determined in phage lambda by the orientation of the packaging origin, cos. cos is a double-chain cut site which serves as a preferred entry site for RecBCD. Using partially denaturing gels to resolve heteroduplex molecules, we have examined patch recombinants at the lambda ren gene. We report that the transferred information in Chi-stimulated patches at ren can occur on either chain, but is biased to the chain ending 5' at the right of the lambda map (the lambda r chain) in phage carrying cos in its normal orientation. The chain bias switches in favor of the chain that ends 3' at the right (the lambda l chain) when RecBCD travel direction is reversed by inverting cos. We entertain models that accommodate these and other results pertaining to the structure of RecBCD-mediated recombinants.
Abstract: RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.
Abstract: Chi is a hotspot for homologous recombination mediated by the RecBCD (Rec) pathway of Escherichia coli. For Rec-mediated recombination of phage lambda, the orientation of lambda cos in the lambda chromosome dictates the direction of travel of RecBCD enzyme through DNA and dictates which orientation of Chi or Chi-like sequences will be active in stimulating recombination. I previously found that Rec-mediated lambda patch heteroduplexes, stimulated by Chi or not, are chain-biased; at the lambda P locus, recombinant information resides on the lambda r chain. This bias exists in the presence or absence of Chi sites. Reported herein is the finding that r-chain-bias at the P locus is independent of the orientation of lambda cos and thus also independent of the orientation of active Chi's or Chi-like sequences and of the direction of travel of RecBCD enzyme. These results disprove previously elaborated models in which a chain-specific nick at Chi initiates recombination, and imply that some other chain-distinguishing process is involved with recombination. Replication and transcription are candidates for such a process.
Abstract: Generalized recombination in Escherichia coli is elevated near Chi sites. In vitro, RecBCD enzyme can nick Chi a few nucleotides 3' of the terminal GG of the Chi sequence (5'-GCTGGTGG). The simplest model in which this nick at Chi participates in Chi function predicts that in phage lambda, Chi-stimulated recombinants not crossed-over for flanking markers (patches) should be heteroduplex, with recombinant information on the lambda I chain. I report here that patches are heteroduplex, but that recombinant information occurs primarily on the lambda r chain. This result rules out the simplest model in which the nick at Chi promotes initiation of recombination, forces reconsideration of Chi's role in recombination, and bears on molecular models for Rec-mediated recombination.
Abstract: In previous systems for in vitro packaging of lambda DNA, phages are produced from the packaging components as well as from added DNA. We have developed a new genetic strategy for in vitro packaging that bypasses this endogenous phage problem. Our system employs a single bacterial strain whose lambda prophage codes for all of the packaging proteins but is deleted for cos, the packaging origin. Crude extracts of the single lysogen: (i) are virtually free from endogenous phages, (ii) package added lambda DNA efficiently and (iii) are easy to prepare. Using the cos- in vitro packaging system we show that packaging of lambda linear monomers is a second-order reaction, but that packaging from concatemers prepared by annealing or ligation is first order. We conclude that in our cos- system, linear monomers are a poor substrate for in vitro packaging but that packaging from concatemers works well.
Abstract: The K restriction system of Escherichia coli works in vitro [Meselson and Yuan, Nature 217 (1968) 1110-1114]. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K-12-derived systems effects a loss of plaque-former output from K-unmodified lambda DNA relative to K-modified lambda DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-175]. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some lambda and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.
