On the Mechanism of Gene Amplification Induced under Stress in Escherichia coli

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. A common change in genomes of all organisms is the reiteration of segments of DNA to multiple copies. DNA amplification can allow rapid evolution by changing the amounts of proteins made, and is instrumental in cancer formation, variation between human genomes, and antibiotic resistance and pathogenicity in microbes. Yet little is known about how amplification occurs, even in simple organisms. DNA amplification can occur in response to stress. In Escherichia coli bacteria, starvation stress provokes amplifications that can allow E. coli ultimately to adjust to the starvation condition. This study elucidates several aspects of the mechanism underlying these stress-provoked amplifications. The data suggest a new model in which DNA replication stalls during starvation, and the end of the new DNA jumps to another stalled replication fork to create a duplicated DNA segment. The duplication can then amplify to many copies by genetic recombination. This model, if correct, can explain how stress provokes these genome rearrangements—by replication stalling. The general model may be useful for other long-distance genome rearrangements in many organisms. Stress can cause rapid and profound changes in the genome, some of which can give cells an advantage—this paper helps to explain how.