The Valley-of-Death: reciprocal sign epistasis constrains adaptive trajectories in a constant, nutrient limiting environment.

The fitness landscape is a powerful metaphor for describing the relationship between genotype and phenotype for a population under selection. However, empirical data as to the topography of fitness landscapes are limited, owing to difficulties in measuring fitness for large numbers of genotypes under any condition. We previously reported a case of reciprocal sign epistasis (RSE), where two mutations individually increased yeast fitness in a glucose-limited environment, but reduced fitness when combined, suggesting the existence of two peaks on the fitness landscape. We sought to determine whether a ridge connected these peaks so that populations founded by one mutant could reach the peak created by the other, avoiding the low-fitness "Valley-of-Death" between them. Sequencing clones after 250 generations of further evolution provided no evidence for such a ridge, but did reveal many presumptive beneficial mutations, adding to a growing body of evidence that clonal interference pervades evolving microbial populations.

Uncoupling reproduction from metabolism extends chronological lifespan in yeast.

Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the age-dependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ∼3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained >95% viability, whereas the viability of starving, freely suspended (planktonic) cells decreased to <10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions.

Kinetic models reveal the in vivo mechanisms of mutagenesis in microbes and man.

This review summarizes the evidence indicating that mutagenic mechanisms in vivo are essentially the same in all living cells. Unique metabolic reactions to a particular environmental stress apparently target specific genes for increased rates of transcription and mutation, resulting in higher mutation rates for those genes most likely to solve the problem. Kinetic models which have demonstrated predictive value are described and are shown to simulate mutagenesis in vivo in Escherichia coli, the p53 tumor suppressor gene, and somatic hypermutation. In all three models, direct correlations are seen between mutation frequencies and transcription rates. G and C nucleosides in single-stranded DNA (ssDNA) are intrinsically mutable, and G and C silent mutations in p53 and in VH framework regions provide compelling evidence for intrinsic mechanisms of mutability, since mutation outcomes are neutral and are not selected. During transcription, the availability of unpaired bases in the ssDNA of secondary structures is rate-limiting for, and determines the frequency of mutations in vivo. In vitro analyses also verify the conclusion that intrinsically mutable bases are in fact located in ssDNA loops of predicted stem-loop structures (SLSs).

Evolution of coordinated mutagenesis and somatic hypermutation in VH5.

The VH5 human antibody gene was analyzed using a computer program (mfg) which simulates transcription, to better understand transcription-driven mutagenesis events that occur during "phase 1" of somatic hypermutation. Results show that the great majority of mutations in the non-transcribed strand occur within loops of two predicted high-stability stem-loop structures, termed SLSs 14.9 and 13.9. In fact, 89% of the 2505 mutations reported are within the encoded complementarity-determining region (CDR) and occur in loops of these high-stability structures. In vitro studies were also done and verified the existence of SLS 14.9. Following the formation of SLSs 14.9 and 13.9, a sustained period of transcriptional activity occurs within a window size of 60-70 nucleotides. During this period, the stability of these two SLSs does not change, and may provide the substrate for base exchanges and mutagenesis. The data suggest that many mutable bases are exposed simultaneously at pause sites, allowing for coordinated mutagenesis.

The roles of transcription and genotoxins underlying p53 mutagenesis in vivo.

Transcription drives supercoiling which forms and stabilizes single-stranded (ss) DNA secondary structures with loops exposing G and C bases that are intrinsically mutable and vulnerable to non-enzymatic hydrolytic reactions. Since many studies in prokaryotes have shown direct correlations between the frequencies of transcription and mutation, we conducted in silico analyses using the computer program, mfg, which simulates transcription and predicts the location of known mutable bases in loops of high-stability secondary structures. Mfg analyses of the p53 tumor suppressor gene predicted the location of mutable bases and mutation frequencies correlated with the extent to which these mutable bases were exposed in secondary structures. In vitro analyses have now confirmed that the 12 most mutable bases in p53 are in fact located in predicted ssDNA loops of these structures. Data show that genotoxins have two independent effects on mutagenesis and the incidence of cancer: Firstly, they activate p53 transcription, which increases the number of exposed mutable bases and also increases mutation frequency. Secondly, genotoxins increase the frequency of G-to-T transversions resulting in a decrease in G-to-A and C mutations. This precise compensatory shift in the 'fate' of G mutations has no impact on mutation frequency. Moreover, it is consistent with our proposed mechanism of mutagenesis in which the frequency of G exposure in ssDNA via transcription is rate limiting for mutation frequency in vivo.

I. VH gene transcription creates stabilized secondary structures for coordinated mutagenesis during somatic hypermutation.

During the adaptive immune response, antigen challenge triggers a million-fold increase in mutation rates in the variable-region antibody genes. The frequency of mutation is causally and directly linked to transcription, which provides ssDNA and drives supercoiling that stabilizes secondary structures containing unpaired, intrinsically mutable bases. Simulation analysis of transcription in VH5 reveals a dominant 65nt secondary structure in the non-transcribed strand containing six sites of mutable ssDNA that have also been identified independently in human B cell lines and in primary mouse B cells. This dominant structure inter-converts briefly with less stable structures and is formed repeatedly during transcription, due to periodic pauses and backtracking. In effect, this creates a stable yet dynamic "mutability platform" consisting of ever-changing patterns of unpaired bases that are simultaneously exposed and therefore able to coordinate mutagenesis. Such a complex of secondary structures may be the source of ssDNA for enzyme-based diversification, which ultimately results in high affinity antibodies.

II. Correlations between secondary structure stability and mutation frequency during somatic hypermutation.

The role of secondary structures and base mutability at different levels of transcription and supercoiling is analyzed in variable region antibody genes VH5, VH94 and VH186.2. The data are consistent with a model of somatic hypermutation in which increasing levels of transcription and secondary structure stability correlate with the initial formation of successive mutable sites. Encoded differences exist in stem length and the number of GC pairs at low versus high levels of transcription in CDRs. These circumstances simplify the complexities of coordinating mutagenesis by confining this process to each mutable site successively, as they form in response to increasing levels of transcription during affinity maturation.

Secondary structures as predictors of mutation potential in the lacZ gene of Escherichia coli.

Four independent nonsense mutations were engineered into the Escherichia coli chromosomal lacZ gene, and reversion rates back to LacZ(+) phenotypes were determined. The mutation potential of bases within putative DNA secondary structures formed during transcription was predicted by a sliding-window analysis that simulates successive folding of the ssDNA creating these structures. The relative base mutabilities predicted by the mfg computer program correlated with experimentally determined reversion rates in three of the four mutants analysed. The nucleotide changes in revertants at one nonsense codon site consisted of a triple mutation, presumed to occur by a templated repair mechanism. Additionally, the effect of supercoiling on mutation was investigated and, in general, reversion rates increased with higher levels of negative supercoiling. Evidence indicates that predicted secondary structures are in fact formed in vivo and that directed mutation in response to starvation stress is dependent upon the exposure of particular bases, the stability of the structures in which these bases are unpaired and the level of DNA supercoiling within the cell.

The effect of promoter strength, supercoiling and secondary structure on mutation rates in Escherichia coli.

Four mutations resulting in opal stop codons were individually engineered into a plasmid-borne chloramphenicol-resistance (cat) gene driven by the lac promoter. These four mutations were located at different sites in secondary structures. The mutations were analysed with the computer program mfg, which predicted their relative reversion frequencies. Reversion frequencies determined experimentally correlated with the mutability of the bases as predicted by mfg. To examine the effect of increased transcription on reversion frequencies, the lac promoter was replaced with the stronger tac promoter, which resulted in 12- to 30-fold increases in reversion rates. The effect of increased and decreased supercoiling was also investigated. The cat mutants had higher reversion rates in a topA mutant strain with increased negative supercoiling compared with wild-type levels, and the cat reversion rates were lower in a topA gyrB mutant strain with decreased negative supercoiling, as predicted.

Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs.

Escherichia coli auxotrophs of leuB and argH were examined to determine if higher rates of transcription in derepressed genes were correlated with increased reversion rates. Rates of leuB and argH mRNA synthesis were determined using half-lives and concentrations, during exponential growth and at several time points during 30 min of amino acid starvation. Changes in mRNA concentration were primarily due to increased mRNA synthesis and not to increased stability. Four strains of E. coli amino acid auxotrophs, isogenic except for relA and argR, were examined. In both the leuB and argH genes, rates of transcription and mutation were compared. In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2 mutants). argR knockout strains were constructed in relA(+) and relA mutant strains, and rates of both argH reversion and mRNA synthesis were significantly higher in the argR knockouts than in the regulated strains. A statistically significant linear correlation between increased rates of transcription and mutation was found for data from both genes. In general, changes in mRNA half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often two orders of magnitude. The results suggest that specific starvation conditions target the biosynthetic genes for derepression and increased rates of transcription and mutation.

Predicting mutation frequencies in stem-loop structures of derepressed genes: implications for evolution.

This work provides evidence that, during transcription, the mutability (propensity to mutate) of a base in a DNA secondary structure depends both on the stability of the structure and on the extent to which the base is unpaired. Zuker's DNA folding computer program reveals the most probable stem-loop structures (SLSs) and negative energies of folding (-DeltaG) for any given nucleotide sequence. We developed an interfacing program that calculates (i) the percentage of folds in which each base is unpaired during transcription; and (ii) the mutability index (MI) for each base, expressed as an absolute value and defined as -follows: MI = (% total folds in which the base is unpaired) x (highest -DeltaG of all folds in which it is unpaired). Thus, MIs predict the relative mutation or reversion frequencies of unpaired bases in SLSs. MIs for 16 mutable bases in auxotrophs, selected during starvation in derepressed genes, are compared with 70 background mutations in lacI and ebgR that were not derepressed during mutant selection. All the results are consistent with the location of known mutable bases in SLSs. Specific conclusions are: (i) Of 16 mutable bases in transcribing genes, 87% have higher MIs than the average base of the sequence analysed, compared with 50% for the 70 background mutations. (ii) In 15 of the mutable bases of transcribing genes, the correlation between MIs and relative mutation frequencies determined experimentally is good. There is no correlation for 35 mutable bases in the lacI gene. (iii) In derepressed auxotrophs, 100% of the codons containing the mutable bases are within one codon's length of a stem, compared with 53% for the background mutable bases in lacI. (iv) The data suggest that environmental stressors may cause as well as select mutations in derepressed genes. The implications of these results for evolution are discussed.

Hypermutable bases in the p53 cancer gene are at vulnerable positions in DNA secondary structures.

A DNA folding analysis indicates that the most hypermutable bases in exons 5, 7, and 8 of the p53 tumor suppressor gene are located immediately next to stems in stable DNA stem-loop structures. On the basis of the highest negative energy (-DeltaG) value of the structures containing each mutable bases and on the extent to which each base is unpaired during transcription, their relative mutabilities are calculated using a new computer algorithm. These predicted mutation frequencies correlate well with those observed in 14,000 human cancers (R(2) = 0.76), whereas there is no such correlation (R(2) = 0.0005) for nearby control bases. The correlation of hypermutable base frequencies with -DeltaG values is poor (R(2) = 0.19), indicating that the extent to which a base is unpaired during transcription is a significant contribution to predicting mutation frequencies.