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.

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.

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.

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.

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.

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 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.

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.

Stress-directed adaptive mutations and evolution.

Comparative biochemistry demonstrates that the metabolites, complex biochemical networks, enzymes and regulatory mechanisms essential to all living cells are conserved in amazing detail throughout evolution. Thus, in order to evolve, an organism must overcome new adverse conditions without creating different but equally dangerous alterations in its ongoing successful metabolic relationship with its environment. Evidence suggests that stable long-term acquisitive evolution results from minor increases in mutation rates of genes related to a particular stress, with minimal disturbance to the balanced and resilient metabolism critical for responding to an unpredictable environment.

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).

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.

Reversion rates in a leuB auxotroph of Escherichia coli K-12 correlate with ppGpp levels during exponential growth.

Two isogenic strains of Escherichia coli K-12 differing only in relA, as well as two spoT transductants of the relA- strain, were examined with respect to ppGpp levels and reversion rates of a leuB- allele under nine different conditions. A positive correlation was established between reversion rates and the steady-state concentration of ppGpp during exponential growth. The leuB genes from two leuB- strains (isogenic except for relA) were cloned and sequenced and found to contain a single mutation, namely, a C-to-T transition at nucleotide 857.