Single-molecule studies of the replisome Visualisation of protein dynamics in multi-protein complexes

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

When proteins play tag: the dynamic nature of the replisome

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

Design of customizable long linear DNA substrates with controlled end modifications for single-molecule studies

2019 Many strategies have been developed to manipulate DNA molecules and investigate protein-DNA interactions with single-molecule resolution. Often, these require long DNA molecules with a length of several 10s of kb that are chemically modified at specific regions. This need has traditionally been met by commercially available DNA from bacteriophage λ. However, λ DNA does not allow for the generation of highly customizable substrates in a straightforward manner, an important factor when developing assays to study complex biochemical reactions. Here we present a generalizable method for the design and production of very long chemically modified DNA substrates derived from a single plasmid. We show the versatility of this design by demonstrating its application in studying DNA replication in vitro. We anticipate this strategy will be broadly useful in producing a range of long chemically modified DNA molecules required for a diverse range of single-molecule approaches

Recycling of single-stranded DNA-binding protein by the bacterial replisome

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

Development of a single-stranded DNA-binding protein fluorescent fusion toolbox

© 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

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

2019 Elsevier Inc. Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α-primase DNA polymerase activity in replication and show that Pol α-primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes