Abstract
Gene amplification mutations occur when a DNA segment is copied numerous times within the genome. These types of mutations can alter gene expression and thus have important implications for evolution. They have been observed across all domains of life and are known drivers of cancer formation in humans and antibiotic resistance in pathogenic bacteria. Despite the biological significance of gene amplification mutations, the mechanisms underlying their formation and collapse remain largely unknown in all organisms. Decades of research have provided a foundation to develop a model for gene amplification. Specifically, this study builds upon the groundwork of an observed relationship between exogenous DNA damage and gene amplification. A bacterial model system in Acinetobacter baylyi that exclusively selects for de novo gene amplification mutants was used to investigate if and how endogenous sources of DNA damage and their repair pathways contribute to gene amplification formation. The first intracellular DNA-damaging agent examined were transposable elements (IS), which are genetic sequences that can physically move throughout the genome. Removing some or all transposable element (IS1236) copies from the A. baylyi genome points to a poignant role of IS elements in gene amplification formation in both parent strains evaluated. Interestingly, these two parent strains only differed genotypically by a single point mutation but displayed distinct manners of IS-mediated gene amplification. The second, less well-characterized DNA-damaging agent explored were R-loops, or DNA-RNA hybrid structures that can form during the transcription process. Examining gene amplification frequencies in various experimental conditions aimed to stimulate R-loops showed a promising connection between them and gene amplification generation. Finally, the major homology-dependent DNA repair systems in A. baylyi (RecA, RecFOR, and RecBCD recombination proteins) were analyzed in-depth to characterize their role in forming gene amplification mutations. These assays illustrated the significant, multifaceted, and overlapping roles of these proteins in DNA repair processes, including those that result in gene amplification. Together, these findings establish a unifying link between endogenous sources of DNA damage and gene amplification and implicate the RecBC-dependent homologous recombination DNA repair pathway as being a major catalyst of gene amplification formation. This study highlights the complex and dynamic nature of gene amplifications and raises important questions about the role of the key players identified and those that remain undiscovered.