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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the replisome. Recent developments in single-molecule approaches have led to a revised picture of the replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.

Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the replisome. Recent developments in single-molecule approaches have led to a revised picture of the replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.

DNA replication, or the copying of DNA, is a fundamental process to all life. The system of proteins that carries out replication, the replisome, encounters many roadblocks on its way. An inability of the replisome to properly overcome these roadblocks will negatively affect genomic integrity which in turn can lead to disease. Over the past decades, efforts by many researchers using a broad array of approaches have revealed roles for many different proteins during the initial response of the replisome upon encountering roadblocks. Here, we revisit what is known about DNA replication and the effect of roadblocks during DNA replication across different organisms. We also address how advances in single-molecule techniques have changed our view of the replisome from a highly stable machine with behavior dictated by deterministic principles to a dynamic system that is controlled by stochastic processes. We propose that these dynamics will play crucial roles in roadblock bypass. Further single-molecule studies of this bypass will, therefore, be essential to facilitate the in-depth investigation of multi-protein complexes that is necessary to understand complicated collisions on the DNA.

DNA replication, or the copying of DNA, is a fundamental process to all life. The system of proteins that carries out replication, the replisome, encounters many roadblocks on its way. An inability of the replisome to properly overcome these roadblocks will negatively affect genomic integrity which in turn can lead to disease. Over the past decades, efforts by many researchers using a broad array of approaches have revealed roles for many different proteins during the initial response of the replisome upon encountering roadblocks. Here, we revisit what is known about DNA replication and the effect of roadblocks during DNA replication across different organisms. We also address how advances in single-molecule techniques have changed our view of the replisome from a highly stable machine with behavior dictated by deterministic principles to a dynamic system that is controlled by stochastic processes. We propose that these dynamics will play crucial roles in roadblock bypass. Further single-molecule studies of this bypass will, therefore, be essential to facilitate the in-depth investigation of multi-protein complexes that is necessary to understand complicated collisions on the DNA.

DNA replication, or the duplication of parental double-stranded DNA (dsDNA) into a pair of identical copies, is essential to the transmission of hereditary information from cell to cell and thus the propagation of all life. It is a fundamental cellular process that is carried out by a multi-protein complex known as the replisome. Since the identification of the first replication proteins by Arthur Kornberg in the 1950s, ensemble-averaging biochemical techniques have been successfully used to study the roles of the various proteins within the replisome. However, the coordination of the multiple activities within the replisome involves transient intermediates and dynamic conformational changes that are difficult, if not impossible, to observe with ensemble experiments. Recently, new single-molecule techniques have been developed to study the dynamics of proteins with a high precision and without the need for population averaging. This thesis centers on the use of these approaches to study the dynamic behaviour of the replisome.