Single-molecule DNA repair in live bacteria

Abstract

Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.single-molecule tracking | super-resolution microscopy | DNA damage response | protein-DNA interaction | cytosolic diffusion A ll cellular organisms rely on complex DNA repair mechanisms for faithful chromosome replication and maintenance of their genome integrity (1). The variety of DNA lesions requires modular repair pathways that carry out damage recognition, damage removal, repair synthesis, and ligation in sequential steps catalyzed by a series of enzymes. However, all repair pathway steps need to be precisely balanced to avoid accumulation of DNA intermediates that are typically more mutagenic and toxic than the original lesion (2). Rapid processing of gapped and nicked intermediates is particularly crucial (3) because they provoke lethal double-strand breaks upon encountering replication forks (4); a single such break can lead to chromosome loss and cell death.Despite extensive genetic, biochemical, and biophysical studies (1), the molecular organization of DNA repair in vivo remains unclear. Most of our mechanistic understanding relies on in vitro ensemble studies, which cannot replicate the cellular environment and stochastic nature of chemical reactions. By avoiding ensembleaveraging, single-molecule experiments have revolutionized the study of protein-DNA interactions in vitro, but extension of these powerful concepts to DNA repair measurements in living cells remains an open goal. Early in vivo work focused on the mean behavior of cell populations and could not examine functionally important heterogeneity, such as the variation in protein copy numbers between cells and over time (5, 6). Such variation can lead to different repair rates across genetically identical cells and may dera...

Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells

“…S4). Referring to other DNA-binding proteins of comparable sizes (33,34), the range of PolC-PAmCherry diffusion coefficients indicates that the slower moving PolC proteins are engaged in confined motion, and we interpret the slow moving PolCPAmCherry as a subpopulation actively engaged in DNA replication.…”

Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid

“…In this technique, trajectories are constructed by determining and connecting the positions of individual particles from consecutive time-lapse images. Importantly, such trajectories can be used to determine whether an individual particle is bound or free if the free particle diffuses significantly faster than its binding targets and remains bound or free for a long time (8,9). Recent advances have made it possible to track hundreds of particles in each cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time (10,11).…”

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

“…The resulting Pol1 localizations occupy the central area of the cell (Figure 6A), broadly recapitulating the spatial organization of the E. coli nucleoid 7 . The majority of Pol1 tracks in undamaged cells display diffusion as shown in Figure 6C.…”