Resolving stalled replication forks in Escherichia coli

DNA replication is essential to successful cell proliferation. Inheritance of traits during cell propagation relies on the accurate duplication of the parental double-stranded DNA (dsDNA) to form two identical daughter copies. This process is carried out by a multi-protein complex referred to as the replisome. Decades of investigations using the model Escherichia coli (E. coli) replisome have provided an overall picture of the process of DNA replication initiation, elongation and termination. However, DNA replication in cells occurs on template DNA coated in DNA-binding proteins that can act as roadblocks and stall the replisome, often resulting in drastic effects on the chromosome. However, the fate of the replisome at these sites remains poorly understood. Stalled DNA replication has been linked to the emergence of antimicrobial resistance in prokaryotes, and the development of severe physical disorders and diseases in eukaryotes. Therefore, understanding the underlying mechanisms of stalled DNA replication can inform future investigations into the maintenance of genome integrity. This thesis focuses on the development and use of single-molecule tools to investigate stalled replication and the resolution of protein roadblocks. Single-molecule tools provide the ability to watch one molecule at a time. Extensive use of these techniques has revealed the heterogeneity that exists within complex biological pathways. Specifically, this thesis highlights the myriad of previously unknown behaviors of proteins on DNA as revealed by single-molecule tools

Nuclease dead Cas9 is a programmable roadblock for DNA replication

Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein-DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9-guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro

Spy-ing on Cas9: Single-molecule tools reveal the enzymology of Cas9

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are an adaptive immune response mechanism in prokaryotes which can target and cleave invading DNA or RNA. The rapid understanding of the type II CRISPR/Cas9 system through biochemical, genetic and structural investigations has contributed to the development of Cas9 for various DNA- and RNA-targeting applications. Recent single-molecule investigations of CRISPR/Cas9 mechanisms have further extended our understanding of target search, binding and cleavage. These investigations are fundamental to the further development of CRISPR/Cas9 tools. This review discusses how single-molecule techniques have illuminated the enzymology of Cas9 endonucleases

Single-molecule studies of helicases and translocases in prokaryotic genome-maintenance pathways

Helicases involved in genomic maintenance are a class of nucleic-acid dependent ATPases that convert the energy of ATP hydrolysis into physical work to execute irreversible steps in DNA replication, repair, and recombination. Prokaryotic helicases provide simple models to understand broadly conserved molecular mechanisms involved in manipulating nucleic acids during genome maintenance. Our understanding of the catalytic properties, mechanisms of regulation, and roles of prokaryotic helicases in DNA metabolism has been assembled through a combination of genetic, biochemical, and structural methods, further refined by single-molecule approaches. Together, these investigations have constructed a framework for understanding the mechanisms that maintain genomic integrity in cells. This review discusses recent single-molecule insights into molecular mechanisms of prokaryotic helicases and translocases

Single-Molecule Fluorescence Imaging of DNA Replication Stalling at Sites of Nucleoprotein Complexes

DNA replication in cells occurs on crowded and often damaged template DNA, forming potentially deleterious roadblocks to the progressing replication fork. Numerous tools have been developed to investigate the mechanisms of DNA replication and the fate of stalled replication forks. Here, we describe single-molecule fluorescence imaging methods to visualize processive DNA replication and replication fork stalling at site-specific nucleoprotein complexes. Using dCas9 as a protein barrier and rolling-circle DNA templates, we visualize effective, stable, and site-specific blocking of the Escherichia coli replisome. Additionally, we present a protocol to produce an 18-kb rolling-circle DNA template that provides increased spatial resolution in imaging the interplay between replisomes and roadblocks. These methods can be used to investigate encounters of the replisome with nucleoprotein complexes at the single-molecule level, providing important mechanistic details of replisome stalling and downstream rescue or restart pathways

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase

Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue