Jacqueline M. Reimers

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. Zukers DNA folding computer program reveals the most probable stem–loop structures (SLSs) and negative energies of folding (–ΔG) 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) × (highest –ΔG 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 codons 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.

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.

Purification and Characterization of the 2-Oxoglutarate Dehydrogenase Complex from Dictyostelium discoideum

The 2-oxoglutarate dehydrogenase complex was isolated from the cellular slime mould, Dictyostelium discoideum, and purified 113-fold. The enzyme exhibited Michaelis-Menten kinetics and the Km values for 2-oxoglutarate, CoA, and NAD were 1.0 mM, 0.002 mM, and 0.07 mM, respectively. The Ki value for succinyl-CoA was determined to be 0.004 mM and the Ki for NADH was 0.018 mM. AMP had positive effects whereas ATP had negative effects on the enzyme activity. The kinetic constants determined in this study and the reaction mechanism suggested can now be incorporated into a transition model of the tricarboxylic acid cycle during differentiation of D. discoideum.

Mechanisms of genotoxin-induced transcription and hypermutation in p53

It is widely assumed that genotoxin-induced damage (e.g., G-to-T transversions) to the tumor suppressor gene, p53, is a direct cause of cancer. However, genotoxins also induce the stress response, which upregulates p53 transcription and the formation of secondary structures from ssDNA. Since unpaired bases are thermodynamically unstable and intrinsically mutable, increased transcription could be the cause of hypermutation, and thus cancer. Support for this hypothesis has been obtained by analyzing 6662 mutations in all types of cancer compared to lung and colon cancers, using the p53 mutation database. The data suggest that genotoxins have two independent effects: first, they induce p53 transcription, which increases the number of mutable bases that determine the incidence of cancer. Second, genotoxins may alter the fate, or ultimate mutation of a mutable base, for example, by causing more of the available mutable Gs to mutate to T, leaving fewer to mutate to A. Such effects on the fate of mutable bases have no impact on the incidence of cancer, as both types of mutations lead to cancer.