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.
Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I-SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.
Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σE acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ32, which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that σE promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by σE, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.
Highlights d Orthogonal DNA sequencing approaches are required to observe all variant types d De novo SNVs and indels accompany non-recurrent structural variants (SVs) in cis d SV-associated SNVs primarily occur within genes and over megabase-sized distances d MMBIR involves extensive DNA replication resulting in regional hypermutation
Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at ∼200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB ∼10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.
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