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.

Multiple pathways of selected gene amplification during adaptive mutation

“…The two colony types described here have been reported previously in both E. coli (19) and S. enterica (9,20). Amplifications of the SJ type have been described in E. coli (21). In S. enterica, the two colony types show a less extreme ratio of stable to unstable Lac ϩ cell types, making it easier to identify the minority cell type.…”

“…The identical nonindependent SJ junctions found in revertants from the first culture could not be detected by PCR in that culture, consistent with their rarity. Identical nonindependent SJ junctions were also observed in E. coli (21).…”

The tandem duplicator phenotype as a distinct genomic configuration in cancer

“…Koszul et al (45) and Koszul and coworkers (46) identified an MH-mediated POL32-dependent replicative mechanism underlying segmental TD formation in S. cerevisiae. A genetic analysis of MH-mediated TD formation in Escherichia coli by Slack et al (47) implicated stalled replication as a trigger to the formation of these TDs. These observations are today collectively termed MMBIR (13,14).…”

Natural Diversity in Flowering Responses of Arabidopsis thaliana Caused by Variation in a Tandem Gene Array

“…Gene duplicates can be found either as dispersed copies or in tandem arrays. While dispersed copies of gene duplicates arise commonly from segmental or whole genome duplication events or retroposition, tandem duplicates can be initiated from single-copy genes by repair of double-stranded DNA breakage and multiplied by unequal crossover events (Sturtevant 1925;Jelesko et al 1999;Blanc et al 2003;Soltis and Soltis 2003;Narayanan et al 2006;Slack et al 2006;Yandeau-Nelson et al 2006;Kong et al 2007;Freeling et al 2008).…”