Detection of kinetic change points in piece-wise linear single molecule motion

Single-molecule approaches present a powerful way to obtain detailed kinetic information at the molecular level. However, the identification of small rate changes is often hindered by the considerable noise present in such single-molecule kinetic data. We present a general method to detect such kinetic change points in trajectories of motion of processive single molecules having Gaussian noise, with a minimum number of parameters and without the need of an assumed kinetic model beyond piece-wise linearity of motion. Kinetic change points are detected using a likelihood ratio test in which the probability of no change is compared to the probability of a change occurring, given the experimental noise. A predetermined confidence interval minimizes the occurrence of false detections. Applying the method recursively to all sub-regions of a single molecule trajectory ensures that all kinetic change points are located. The algorithm presented allows rigorous and quantitative determination of kinetic change points in noisy single molecule observations without the need for filtering or binning, which reduce temporal resolution and obscure dynamics. The statistical framework for the approach and implementation details are discussed. The detection power of the algorithm is assessed using simulations with both single kinetic changes and multiple kinetic changes that typically arise in observations of single-molecule DNA-replication reactions. Implementations of the algorithm are provided in ImageJ plugin format written in Java and in the Julia language for numeric computing, with accompanying Jupyter Notebooks to allow reproduction of the analysis presented here

Single-molecule visualization of fast polymerase turnover in the bacterial replisome

The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment

Structural and Biochemical Studies of Origin Opening by Bacterial Replication Initiators

All organisms depend on a variety of oligomeric ATPase assemblies to carry out essential cellular processes ranging from proteolysis and membrane trafficking to signaling events and nucleic acid transactions. The onset of DNA replication is one such process, relying on dedicated, ATP-dependent initiation factors to coordinate origin recognition and replisome assembly. Although cellular initiators and the initiators of certain classes of double-stranded DNA viruses use a variety of different strategies to process replication origins, they all possess a common adenine nucleotide-binding fold belonging to the AAA+ family of ATPases. To better understand the origin processing mechanism of AAA+ initiators, I performed a series of biochemical and structural studies with the bacterial initiator DnaA. In one study, I used structure-guided mutagenesis, biochemical, and genetic approaches to show that different oligomeric conformations of DnaA play distinct roles in controlling the progression of initiation. In a second study, I showed both crystallographically and in solution that the AAA+ domains of a spiral DnaA oligomer bind and extend single-stranded DNA segments in a RecA-like manner. These structural insights, combined with a novel DNA melting assay, indicate that DnaA uses its AAA+ domain to directly open replication origins by an active, ATP-dependent stretching mechanism. The studies presented in this dissertation provide a new model for origin opening in bacteria that has implications for the origin processing mechanism of all initiators

Structural and Biochemical Studies of Origin Opening by Bacterial Replication Initiators

All organisms depend on a variety of oligomeric ATPase assemblies to carry out essential cellular processes ranging from proteolysis and membrane trafficking to signaling events and nucleic acid transactions. The onset of DNA replication is one such process, relying on dedicated, ATP-dependent initiation factors to coordinate origin recognition and replisome assembly. Although cellular initiators and the initiators of certain classes of double-stranded DNA viruses use a variety of different strategies to process replication origins, they all possess a common adenine nucleotide-binding fold belonging to the AAA+ family of ATPases. To better understand the origin processing mechanism of AAA+ initiators, I performed a series of biochemical and structural studies with the bacterial initiator DnaA. In one study, I used structure-guided mutagenesis, biochemical, and genetic approaches to show that different oligomeric conformations of DnaA play distinct roles in controlling the progression of initiation. In a second study, I showed both crystallographically and in solution that the AAA+ domains of a spiral DnaA oligomer bind and extend single-stranded DNA segments in a RecA-like manner. These structural insights, combined with a novel DNA melting assay, indicate that DnaA uses its AAA+ domain to directly open replication origins by an active, ATP-dependent stretching mechanism. The studies presented in this dissertation provide a new model for origin opening in bacteria that has implications for the origin processing mechanism of all initiators

A structural framework for replication origin opening by AAA plus initiation factors

ATP-dependent initiation factors help process replication origins and coordinate replisome assembly to control the onset of DNA synthesis. Although the specific properties and regulatory mechanisms of initiator proteins can vary greatly between different organisms, certain nucleotide-binding elements and assembly patterns appear preserved not only within the three domains of cellular life (bacteria, archaea, and eukaryotes), but also with certain classes of double-stranded DNA viruses. Structural studies of replication initiation proteins, both as higher-order oligomers and in complex with cognate DNA substrates, are revealing how an evolutionarily related ATPase fold can support different modes of macromolecular assembly and function. Comparative studies between initiation systems in turn provide clues as to how duplex origin regions may be melted during initiation events

Stability versus exchange: A paradox in DNA replication

Multi-component biological machines, comprising individual proteins with specialized functions, perform a variety of essential processes in cells. Once assembled, most such complexes are considered very stable, retaining individual constituents as long as required. However, rapid and frequent exchange of individual factors in a range of critical cellular assemblies, including DNA replication machineries, DNA transcription regulators and flagellar motors, has recently been observed. The high stability of a multi-protein complex may appear mutually exclusive with rapid subunit exchange. Here, we describe a multisite competitive exchange mechanism, based on simultaneous binding of a protein to multiple low-affinity sites. It explains how a component can be stably integrated into a complex in the absence of competing factors, while able to rapidly exchange in the presence of competing proteins. We provide a mathematical model for the mechanism and give analytical expressions for the stability of a pre-formed complex, in the absence and presence of competitors. Using typical binding kinetic parameters, we show that the mechanism is operational under physically realistic conditions. Thus, high stability and rapid exchange within a complex can be reconciled and this framework can be used to rationalize previous observations, qualitatively as well as quantitatively

Replication-fork dynamics

The proliferation of all organisms depends on the coordination of enzymatic events within large multiprotein replisomes that duplicate chromosomes. Whereas the structure and function of many core replisome components have been clarified, the timing and order of molecular events during replication remains obscure. To better understand the replication mechanism, new methods must be developed that allow for the observation and characterization of short-lived states and dynamic events at single replication forks. Over the last decade, great progress has been made toward this goal with the development of novel DNA nanomanipulation and fluorescence imaging techniques allowing for the direct observation of replication-fork dynamics both reconstituted in vitro and in live cells. This article reviews these new single-molecule approaches and the revised understanding of replisome operation that has emerged