An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell. To capture these dynamics, we develop lag-time analysis, a method for measuring in vivo dynamics. Using this approach, we provide quantitative locus-specific measurements of fork velocity, in units of kilobases per second, as well as replisome pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In this work, we quantitatively characterize known phenomena, detect brief, locus-specific pauses at ribosomal DNA loci in wild-type cells, and observe temporal fork velocity oscillations in three highly-divergent bacterial species.

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Antimicrobial resistance (AMR) rapidly develops against almost all available therapeutics. New antibiotics target essential processes in bacteria but fail to address the root of the problem: mutagenesis and subsequent evolution. We recently proposed that inhibiting evolution is the ultimate solution to preventing AMR development. Here, we describe the first compound that inhibits the evolution of AMR by directly targeting a highly conserved evolvability factor, Mfd. We previously found that this RNA polymerase-associated translocase is required for rapid AMR development across highly divergent pathogens. Through an in vivo screen of novel compounds, we identified a small molecule (referred to as ARM-1) that binds Mfd and prevents its RNA polymerase termination activity. Inhibition of Mfd activity by ARM-1 delays the development of mutations, both in pure culture and during infection. Importantly, our data show that ARM-1 prevents the evolution of AMR across highly divergent pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus, which cause extremely difficult to treat infections due to AMR development. In summary, we describe a novel compound that could be developed into a clinically usable anti-evolution drug. This work shows that the mechanisms accelerating evolution are druggable, and that this strategy could prevent AMR development in the clinic.

An important step towards understanding the replication process in the context of the cell is the quantitative characterization of replication dynamics, including the rate of replication fork progression (i.e. fork velocity) with genomic and temporal specificity in vivo. In this paper, we develop a novel method, lag-time analysis, for measuring replisome dynamics using next-generation sequencing. We provide the first quantitative locus-specific measurements of fork velocity. The measured velocity is observed to be both locus and time dependent, even in wild-type cells. To benchmark the approach, we analyze replication dynamics in three different species and a collection of mutants which facilitate the quantitative characterization of replication-conflict-induced fork slowdowns as well as a host of other replication dynamics phenomena, including the observation of temporal fork velocity oscillation. With increases in sequencing depth and improvements in sample preparation, the approach has the potential to provide new insights at higher genomic resolution and in a wide range of biological systems.