Repair on the Go: E. coli Maintains a High Proliferation Rate while Repairing a Chronic DNA Double-Strand Break

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.     

A quasispecies approach to the evolution of sexual replication in unicellular organisms

This study develops a simplified model describing the evolutionary dynamics of a population composed of obligate sexually and asexually reproducing, unicellular organisms. The model assumes that the organisms have diploid genomes consisting of two chromosomes, and that the sexual organisms replicate by first dividing into haploid intermediates, which then combine with other haploids, followed by the normal mitotic division of the resulting diploid into two new daughter cells. We assume that the fitness landscape of the diploids is analogous to the single-fitness-peak approach often used in single-chromosome studies. That is, we assume a master chromosome that becomes defective with just one point mutation. The diploid fitness then depends on whether the genome has zero, one, or two copies of the master chromosome. We also assume that only pairs of haploids with a master chromosome are capable of combining so as to produce sexual diploid cells, and that this process is described by second-order kinetics. We find that, in a range of intermediate values of the replication fidelity, sexually reproducing cells can outcompete asexual ones, provided the initial abundance of sexual cells is above some threshold value. The range of values where sexual reproduction outcompetes asexual reproduction increases with decreasing replication rate and increasing population density. We critically evaluate a common approach, based on a group selection perspective, used to study the competition between populations and show its flaws in addressing the evolution of sex problem.     

Repression/Depression of Conjugative Plasmids and Their Influence on the Mutation-Selection Balance in Static Environments

We study the effect that conjugation-mediated Horizontal Gene Transfer (HGT) has on the mutation-selection balance of a population in a static environment. We consider a model whereby a population of unicellular organisms, capable of conjugation, comes to mutation-selection balance in the presence of an antibiotic, which induces a first-order death rate constant for genomes that are not resistant. We explicitly take into consideration the repression/de-repression dynamics of the conjugative plasmid, and assume that a de-repressed plasmid remains temporarily de-repressed after copying itself into another cell. We assume that both repression and de-repression are characterized by first-order rate constants and , respectively. We find that conjugation has a deleterious effect on the mean fitness of the population, suggesting that HGT does not provide a selective advantage in a static environment, but is rather only useful for adapting to new environments. This effect can be ameliorated by repression, suggesting that while HGT is not necessarily advantageous for a population in a static environment, its deleterious effect on the mean fitness can be negated via repression. Therefore, it is likely that HGT is much more advantageous in a dynamic landscape. Furthermore, in the limiting case of a vanishing spontaneous de-repression rate constant, we find that the fraction of conjugators in the population undergoes a phase transition as a function of population density. Below a critical population density, the fraction of conjugators is zero, while above this critical population density the fraction of conjugators rises continuously to one. Our model for conjugation-mediated HGT is related to models of infectious disease dynamics, where the conjugators play the role of the infected (I) class, and the non-conjugators play the role of the susceptible (S) class.     

Cellular response to horizontally transferred DNA in Escherichia coli is tuned by DNA repair systems

We studied how DNA divergence between recombining DNAs and the mismatch repair system modulate the SOS response in Escherichia coli. The observed positive log-linear correlation between SOS induction and DNA divergence, and the negative correlation between SOS induction and frequency of recombination, suggest that the level of SOS induction precisely reflects the difficulty of RecA protein to initiate a productive strand exchange process. Our results suggest that the mismatch repair system could contribute to this SOS induction more by affecting the RecA-catalyzed homology search than by acting on mismatched recombination intermediates. The propensity of the recombination machinery to promote recombination between the blocks of sequences with the highest identity results in the increasing ratios of merodiploids (partial diploids) over genuine recombinants (homologous replacements) with increasing DNA divergence. We discuss the role of molecular mechanisms involved in the control of the recombination between diverged DNA sequences in the maintenance of genomic stability and genome evolution.     

Selective advantage for sexual reproduction with random haploid fusion

This article develops a simplified set of models describing asexual and sexual replication in unicellular diploid organisms. The models assume organisms whose genomes consist of two chromosomes, where each chromosome is assumed to be functional if it is equal to some master sequence σ₀, and non-functional otherwise. We review the previously studied case of selective mating, where it is assumed that only haploids with functional chromosomes can fuse, and also consider the case of random haploid fusion. When the cost for sex is small, as measured by the ratio of the characteristic haploid fusion time to the characteristic growth time, we find that sexual replication with random haploid fusion leads to a greater mean fitness for the population than a purely asexual strategy. However, independently of the cost for sex, we find that sexual replication with a selective mating strategy leads to a higher mean fitness than the random mating strategy. The results of this article are consistent with previous studies suggesting that sex is favored at intermediate mutation rates, for slowly replicating organisms, and at high population densities. Furthermore, the results of this article provide a basis for understanding sex as a stress response in unicellular organisms such as Saccharomyces cerevisiae (Baker’s yeast).     

Effect of chromosomal instability on the mutation-selection balance in unicellular populations

This paper develops a mathematical model describing the evolutionary dynamics of a unicellular, asexually replicating population exhibiting chromosomal instability. Chromosomal instability is a form of genetic instability characterized by the gain or loss of entire chromosomes during cell division. We assume that the cellular genome is divided into several homologous groups of chromosomes, and that a single functional chromosome per homologous group is required for the cell to have the wild-type fitness. If the fitness is unaffected by the total number of chromosomes in the cell, our model is analytically solvable, and yields a mean fitness at mutation-selection balance that is identical to the mean fitness when there is no chromosomal instability. If this assumption is relaxed and the total number of chromosomes in the cell is not allowed to increase without bound, then chromosomal instability leads to a reduction in mean fitness. The results of this paper provide a useful baseline that can inform both future theoretial and experimental studies of chromosomal instability.     

The SOS response increases bacterial fitness,but not evolvability,under a sublethal dose of antibiotic

Exposure to antibiotics induces the expression of mutagenic bacterial stress–response pathways, but the evolutionary benefits of these responses remain unclear. One possibility is that stress–response pathways provide a short-term advantage by protecting bacteria against the toxic effects of antibiotics. Second, it is possible that stress-induced mutagenesis provides a long-term advantage by accelerating the evolution of resistance. Here, we directly measure the contribution of the Pseudomonas aeruginosa SOS pathway to bacterial fitness and evolvability in the presence of sublethal doses of ciprofloxacin. Using short-term competition experiments, we demonstrate that the SOS pathway increases competitive fitness in the presence of ciprofloxacin. Continued exposure to ciprofloxacin results in the rapid evolution of increased fitness and antibiotic resistance, but we find no evidence that SOS-induced mutagenesis accelerates the rate of adaptation to ciprofloxacin during a 200 generation selection experiment. Intriguingly, we find that the expression of the SOS pathway decreases during adaptation to ciprofloxacin, and this helps to explain why this pathway does not increase long-term evolvability. Furthermore, we argue that the SOS pathway fails to accelerate adaptation to ciprofloxacin because the modest increase in the mutation rate associated with SOS mutagenesis is offset by a decrease in the effective strength of selection for increased resistance at a population level. Our findings suggest that the primary evolutionary benefit of the SOS response is to increase bacterial competitive ability, and that stress-induced mutagenesis is an unwanted side effect, and not a selected attribute, of this pathway.     

Diploidy and the selective advantage for sexual reproduction in unicellular organisms

This article develops mathematical models describing the evolutionary dynamics of both asexually and sexually reproducing populations of diploid unicellular organisms. The asexual and sexual life cycles are based on the asexual and sexual life cycles in Saccharomyces cerevisiae, Baker’s yeast, which normally reproduces by asexual budding, but switches to sexual reproduction when stressed. The mathematical models consider three reproduction pathways: (1) Asexual reproduction, (2) self-fertilization, and (3) sexual reproduction. We also consider two forms of genome organization. In the first case, we assume that the genome consists of two multi-gene chromosomes, whereas in the second case, we consider the opposite extreme and assume that each gene defines a separate chromosome, which we call the multi-chromosome genome. These two cases are considered to explore the role that recombination has on the mutation-selection balance and the selective advantage of the various reproduction strategies. We assume that the purpose of diploidy is to provide redundancy, so that damage to a gene may be repaired using the other, presumably undamaged copy (a process known as homologous recombination repair). As a result, we assume that the fitness of the organism only depends on the number of homologous gene pairs that contain at least one functional copy of a given gene. If the organism has at least one functional copy of every gene in the genome, we assume a fitness of 1. In general, if the organism has l homologous pairs that lack a functional copy of the given gene, then the fitness of the organism is κ _l . The κ _l are assumed to be monotonically decreasing, so that κ₀ = 1 > κ₁ > κ₂ > ⋯ > κ_∞ = 0. For nearly all of the reproduction strategies we consider, we find, in the limit of large N, that the mean fitness at mutation-selection balance is max{2 e^-m-1, 0} ,\hbox{max}\{2 e^{-\mu}-1, 0\} , where N is the number of genes in the haploid set of the genome, ε is the probability that a given DNA template strand of a given gene produces a mutated daughter during replication, and μ = Nε. The only exception is the sexual reproduction pathway for the multi-chromosomed genome. Assuming a multiplicative fitness landscape where κ _l  = α ^l for α ∈ (0, 1), this strategy is found to have a mean fitness that exceeds the mean fitness of all the other strategies. Furthermore, while other reproduction strategies experience a total loss of viability due to the steady accumulation of deleterious mutations once μ exceeds ln2 ,\ln 2 , no such transition occurs in the sexual pathway. Indeed, in the limit as α → 1 for the multiplicative landscape, we can show that the mean fitness for the sexual pathway with the multi-chromosomed genome converges to e ^−2μ, which is always positive. We explicitly allow for mitotic recombination in this study, which, in contrast to previous studies using different models, does not have any advantage over other asexual reproduction strategies. The results of this article provide a basis for understanding the selective advantage of the specific meiotic pathway that is employed by sexually reproducing organisms. The results of this article also suggest an explanation for why unicellular organisms such as Saccharomyces cerevisiae (Baker’s yeast) switch to a sexual mode of reproduction when stressed. While the results of this article are based on modeling mutation-propagation in unicellular organisms, they nevertheless suggest that, in more complex organisms with significantly larger genomes, sex is necessary to prevent the loss of viability of a population due to genetic drift. Finally, and perhaps most importantly, the results of this article demonstrate a selective advantage for sexual reproduction with fewer and much less restrictive assumptions than those of previous studies.     

Division of labour and the evolution of multicellularity

Understanding the emergence and evolution of multicellularity and cellular differentiation is a core problem in biology. We develop a quantitative model that shows that a multicellular form emerges from genetically identical unicellular ancestors when the compartmentalization of poorly compatible physiological processes into component cells of an aggregate produces a fitness advantage. This division of labour between the cells in the aggregate occurs spontaneously at the regulatory level owing to mechanisms present in unicellular ancestors and does not require any genetic predisposition for a particular role in the aggregate or any orchestrated cooperative behaviour of aggregate cells. Mathematically, aggregation implies an increase in the dimensionality of phenotype space that generates a fitness landscape with new fitness maxima, in which the unicellular states of optimized metabolism become fitness saddle points. Evolution of multicellularity is modelled as evolution of a hereditary parameter: the propensity of cells to stick together, which determines the fraction of time a cell spends in the aggregate form. Stickiness can increase evolutionarily owing to the fitness advantage generated by the division of labour between cells in an aggregate.     

Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage.

The induction of the SOS response by H2O2 was measured in Escherichia coli by means of a sfiA::lacZ operon fusion. The effects of mutations in genes involved in DNA repair or DNA metabolism on the SOS response were investigated. We found that in an uvrA mutant, H2O2 induced the SOS response at lower concentrations than in the uvr+ parent strain, indicating that some lesions induced by H2O2 may be repaired by the uvrABC-dependent excision repair system. A nth mutation, yielding deficiency in thymine glycol DNA glycosylase, had no detectable effect on SOS induction, indicating that thymine glycol, a DNA lesion expected to be induced by H2O2, does not participate detectably in the induction of the SOS response by this chemical under our conditions. H2O2 still induced the SOS response in a dnaC(Ts) uvrA double mutant under conditions in which no DNA replication proceeds, suggesting that this chemical induces DNA strand breaks. Induction of the SOS response by H2O2 was also assayed in various mutants affected in genes suspected to be important for protection against oxidative stress. Mutations in the catalase genes, katE and katG, had only minor effects. However, in an oxyR deletion mutant, in which the adaptative response to H2O2 does not occur, SOS induction occurred at much lower H2O2 concentrations than in the oxyR+ parent strain. These results indicate that some enzymes regulated by the oxyR gene are, under our conditions, more important than catalase for protection against the H2O2-induced DNA damages which trigger the SOS response.     

Analysis of the SOS inducing signal in Bacillus subtilis using Escherichia coli LexA as a probe.

We analyzed the Bacillus subtilis SOS response using Escherichia coli LexA protein as a probe to measure the kinetics of SOS activation and DNA repair in wild-type and DNA repair-deficient strains. By examining the effects of DNA-damaging agents that produce the SOS inducing signal in E. coli by three distinct pathways, we obtained evidence that the nature of the SOS inducing signal has been conserved in B. subtilis. In particular, we used the B. subtilis DNA polymerase III inhibitor, 6-(p-hydroxyphenylazo)-uracil, to show that DNA replication is required to generate the SOS inducing signal following UV irradiation. We also present evidence that single-stranded gaps, generated by excision repair, serve as part of the UV inducing signal. By assaying the SOS response in B. subtilis dinA, dinB, and dinC mutants, we identified distinct deficiencies in SOS activation and DNA repair that suggest roles for the corresponding gene products in the SOS response.     

UV induction of LexA independent proteins which could be involved in SOS repair

The SOS response is induced in E coli following treatments that interfere with DNA replication. The response is under the control of the recA and the lexA genes. Strains defective in LexA repressor constitutively express SOS proteins. However, SOS repair does not reach its maximum level in these strains. Instead, an activation of RecA protein and de novo protein synthesis are required for full repair. We have analyzed by 2-dimensional gel electrophoresis the induction of proteins after UV irradiation of lexA(Def) bacteria. Proteins which might participate in SOS repair are induced under these conditions.     

Evolution in fine-grained environments I. Environmental runs and the evolution of homeostasis

In this paper we discuss the problem of evolution when individual organisms are subjected to heterogeneous environments within their own lifetimes. We first develop a model of environmental heterogeneity in which there are two discrete environmental states. Transitions between states are governed by a stochastic matrix. Next, we examine how an organism responds to this heterogeneity. We assume that L consecutive time units of the environmental process are sampled during the normal life span of the organism, and that the individual's fitness is determined in part by a component unrelated to this heterogeneity and by other components that describe the fitness response to the heterogeneity. The fitness responses are functions of the environmental state and of how long the organism has been previously exposed to that state; i.e., fitness response is dependent upon the environmental context. We then discuss how this individually experienced heterogeneity is translated to the populational level. Finally, genetic constraints are overlaid so that the tools of population genetics may be used to make evolutionary predictions.     

The Selection-Mutation-Drift Theory of Synonymous Codon Usage

It is argued that the bias in synonymous codon usage observed in unicellular organisms is due to a balance between the forces of selection and mutation in a finite population, with greater bias in highly expressed genes reflecting stronger selection for efficiency of translation. A population genetic model is developed taking into account population size and selective differences between synonymous codons. A biochemical model is then developed to predict the magnitude of selective differences between synonymous codons in unicellular organisms in which growth rate (or possibly growth yield) can be equated with fitness. Selection can arise from differences in either the speed or the accuracy of translation. A model for the effect of speed of translation on fitness is considered in detail, a similar model for accuracy more briefly. The model is successful in predicting a difference in the degree of bias at the beginning than in the rest of the gene under some circumstances, as observed in Escherichia coli, but grossly overestimates the amount of bias expected. Possible reasons for this discrepancy are discussed.     

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.     

Localization of UvrA and Effect of DNA Damage on the Chromosome of Bacillus subtilis

We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells. We suggest that the UvrA(2)B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response. We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell's genome. Similar observations have been made in Escherichia coli, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.     

Simulating the temporal modulation of inducible DNA damage response in Escherichia coli

Living organisms make great efforts to maintain their genetic information integrity. However, DNA is vulnerable to many chemical or physical agents. To rescue the cell timely and effectively, the DNA damage response system must be well controlled. Recently, single cell experiments showing that after DNA damage, expression of the key DNA damage response regulatory protein oscillates with time. This phenomenon is observed both in eukaryotic and bacterial cells. We establish a model to simulate the DNA damage response (SOS response) in bacterial cell Escherichia coli. The simulation results are compared to the experimental data. Our simulation results suggest that the modulation observed in the experiment is due to the fluctuation of inducing signal, which is coupled with DNA replication. The inducing signal increases when replication is blocked by DNA damage and decreases when replication resumes.     

Evolution of DNA double-strand break repair by gene conversion: coevolution between a phage and a restriction-modification system

The necessity to repair genome damage has been considered to be an immediate factor responsible for the origin of sex. Indeed, attack by a cellular restriction enzyme of invading DNA from several bacteriophages initiates recombinational repair by gene conversion if there is homologous DNA. In this work, we modeled the interaction between a bacteriophage and a bacterium carrying a restriction enzyme as antagonistic coevolution. We assume a locus on the bacteriophage genome has either a restriction-sensitive or a restriction-resistant allele, and another locus determines whether it is recombination/repair proficient or defective. A restriction break can be repaired by a co-infecting phage genome if one of them is recombination/repair proficient. We define the fitness of phage (resistant/sensitive and repair-positive/-negative) genotypes and bacterial (restriction-positive/-negative) genotypes by assuming random encounter of the genotypes, with given probabilities of single and double infections, and the costs of resistance, repair, and restriction. Our results show the evolution of the repair allele depends on b(1)/b(0), the ratio of the burst size b(1) under damage to host cell physiology induced by an unrepaired double-strand break to the default burst size b(0). It was not until this effect was taken into account that the evolutionary advantage of DNA repair became apparent.     

Inducible DNA repair and differentiation in Bacillus subtilis: interactions between global regulons

The SOS response of Escherichia coli has become a paradigm for the study of inducible DNA repair and recombination processes in many different organisms. While these studies have demonstrated that the components of the SOS response appear to be highly conserved among bacterial species, as with most models, there are some significant variations. Perhaps the best example of this comes from an analysis of the SOS-like system of the developmental organism, Bacillus subtilis. Accordingly, the most striking difference is the complex developmental regulation of the SOS system as this organism differentiates into its competent state. In this review we have given an overview of the elements that comprise the SOS system of B. subtilis. Additionally, we have summarized our most recent findings regarding the regulation of this regulon. Using these results along with new findings from other laboratories we have provided provocative molecular models for the regulation of the B. subtilis SOS system in response to DNA damage and during competent cell formation.