Single-molecule Sorting Research Articles

Single-Molecule Sorting of Human DNA Repair Enzymes

Functional diversity and the dynamic nature of eukaryotic proteome are highly enhanced by posttranslational modifications. Activities of human DNA repair helicases are often controlled by phosphorylation, sumoylation and ubiquitylation. These posttranslational modifications may directly influence the helicase activity or may direct the helicase integration into a particular molecular machine. It is foreseeable that in some cases a few or even a single protein molecule carrying a particular array of modifications could decide the fate of the cell. The presence of a heterogeneous helicase pool in the cell complicates the helicase's biochemical analysis and its structure activity understanding. To this end we have developed single-molecule sorting to quantify, simultaneously probe and distinguish activities of DNA helicases carrying the posttranslational modifications. We surface-tether in vivo biotinylated helicase molecules expressed in human cells. We then use total internal reflection fluorescence microscopy (TIRFM) to follow activity of each individual surface-tethered molecule over multiple cycles of binding and rearrangement of fluorescently labeled DNA substrates or nucleoprotein complexes. The presence of posttranslational modifications is then revealed and is correlated with the measured activity. The difference between the modified and unmodified enzymes can be evaluated even when only a small fraction of helicase molecules carries the modification of interest. Our experimental strategy overcomes difficulties associated with the comprehensive analysis of heterologously modified cellular pool of proteins. It also circumvents another critical hurdle associated with quantitative analysis of large human proteins, namely the difficulty in obtaining large quantities of active protein. I will discuss application of the single-molecule sorting methodology to study two human DNA repair helicases FBH1 and FANCJ.

Single-molecule sorting of DNA helicases

DNA helicases participate in virtually all aspects of cellular DNA metabolism by using ATP-fueled directional translocation along the DNA molecule to unwind DNA duplexes, dismantle nucleoprotein complexes, and remove non-canonical DNA structures. Post-translational modifications and helicase interacting partners are often viewed as determining factors in controlling the switch between bona fide helicase activity and other functions of the enzyme that do not involve duplex separation. The bottleneck in developing a mechanistic understanding of human helicases and their control by post-translational modifications is obtaining sufficient quantities of the modified helicase for traditional structure-functional analyses and biochemical reconstitutions. This limitation can be overcome by single-molecule analysis, where several hundred surface-tethered molecules are sufficient to obtain a complete kinetic and thermodynamic description of the helicase-mediated substrate binding and rearrangement. Synthetic oligonucleotides site-specifically labeled with Cy3 and Cy5 fluorophores can be used to create a variety of DNA substrates that can be used to characterize DNA binding, as well as helicase translocation and duplex unwinding activities. This chapter describes “single-molecule sorting”, a robust experimental approach to simultaneously quantify, and distinguish the activities of helicases carrying their native post-translational modifications. Using this technique, a DNA helicase of interest can be produced and biotinylated in human cells to enable surface-tethering for the single-molecule studies by total internal reflection fluorescence microscopy. The pool of helicases extracted from the cells is expected to contain a mixture of post-translationally modified and unmodified enzymes, and the contributions from either population can be monitored separately, but in the same experiment providing a direct route to evaluating the effect of a given modification.

FBH1 Catalyzes Regression of Stalled Replication Forks.

DNA replication fork perturbation is a major challenge to the maintenance of genome integrity. It has been suggested that processing of stalled forks might involve fork regression, in which the fork reverses and the two nascent DNA strands anneal. Here, we show that FBH1 catalyzes regression of a model replication fork invitro and promotes fork regression invivo in response to replication perturbation. Cells respond to fork stalling by activating checkpoint responses requiring signaling through stress-activated protein kinases. Importantly, we show that FBH1, through its helicase activity, is required for early phosphorylation of ATM substrates such as CHK2 and CtIP as well as hyperphosphorylation of RPA. These phosphorylations occur prior to apparent DNA double-strand break formation. Furthermore, FBH1-dependent signaling promotes checkpoint control and preserves genome integrity. We propose a model whereby FBH1 promotes early checkpoint signaling by remodeling of stalled DNA replication forks.

Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase

DNA repair helicases function in the cell to separate DNA duplexes or remodel nucleoprotein complexes. These functions are influenced by sensing and signaling; the cellular pool of a DNA helicase may contain subpopulations of enzymes carrying different post-translational modifications and performing distinct biochemical functions. Here, we report a novel experimental strategy, single-molecule sorting, which overcomes difficulties associated with comprehensive analysis of heterologously modified pool of proteins. This methodology was applied to visualize human DNA helicase F-box–containing DNA helicase (FBH1) acting on the DNA structures resembling a stalled or collapsed replication fork and its interactions with RAD51 nucleoprotein filament. Individual helicase molecules isolated from human cells with their native post-translational modifications were analyzed using total internal reflection fluorescence microscopy. Separation of the activity trajectories originated from ubiquitylated and non-ubiquitylated FBH1 molecules revealed that ubiquitylation affects FBH1 interaction with the RAD51 nucleoprotein filament, but not its translocase and helicase activities.

2P289 流体力学的絞込みを用いた一分子ソーターセルの開発(26.計測,ポスター,日本生物物理学会年会第51回(2013年度))

2P289 流体力学的絞込みを用いた一分子ソーターセルの開発(26.計測,ポスター,日本生物物理学会年会第51回(2013年度))

Single-Molecule Sorting of DNA Repair Machines

Often, molecular machines orchestrating distinct types of DNA repair share motor components known as DNA helicases, ubiquitous vectorial enzymes that use chemical energy of ATP to translocate directionally on the DNA, transiently unwind duplex DNA or remodel protein-nucleic acid complexes. Participation in different pathways may require a helicase to switch between different activities (for example, between being a bona fide helicase versus a DNA translocase that dismantles protein-nucleic acid complexes), to down- or up-regulate its processivity or to recognize a different substrate. Activity switching in DNA repair enzymes is often controlled by reversible post-translational modifications. Addition or removal of these modifications may rapidly activate or inactivate one or several activities of a target helicase or may alter its molecular interactions.To address the role of post-translational modification and specific interactions in directing a helicase to a particular genome maintenance event, we developed an experimental system we refer to as single-molecule sorting. This single-molecule total internal reflection microscopy (TIRFM) approach discriminates between subpopulations of differentially modified enzymes and correlates the presence of each modification to the biochemical function and therefore to the biological role of a particular helicase. Application of single-molecule sorting to deciphering the molecular basis of pro- and anti-recombinogenic activity of human DNA repair helicase Fbh1 and its regulation will be discussed.

1PT222 一分子蛍光ソーティングを目指したマイクロ流体システムの構築(日本生物物理学会第50回年会(2012年度))

1PT222 一分子蛍光ソーティングを目指したマイクロ流体システムの構築(日本生物物理学会第50回年会(2012年度))

Nanofluidic single-molecule sorting of DNA: a new concept in separation and analysis ofbiomolecules towards ultimate level performance

Separation and separation-based analysis of biomolecules are fundamentally importanttechniques in the field of biotechnology. These techniques, however, depend onstochastic processes that intrinsically involve uncertainty, and thus it is not possible toachieve 100% separation accuracy. Theoretically, the ultimate resolution andsensitivity should be realized in a single-molecule system because of the deterministicnature of single-molecule manipulation. Here, we have proposed and experimentallydemonstrated the concept of a ‘single-molecule sorter’ that detects and correctly identifiesindividual single molecules, realizing the ultimate level of resolution and sensitivity forany separation-based technology. The single-molecule sorter was created using ananofluidic network consisting of a single inlet channel that branches off intomultiple outlet channels. It includes two major functional elements, namely asingle-molecule detection and identification element and a flow path switching elementto accurately separate single molecules. With this system we have successfullydemonstrated the world’s first single-molecule sorting using DNA as a samplemolecule. In the future, we hope to expand the application of such devices tocomprehensive sorting of single-proteins from a single cell. We also believe thatin addition to the single-molecule sorting method reported here, other types ofsingle-molecule based processes will emerge and find use in a wide variety of applications.

Analytic Solution to the Autocorrelation Function for Lateral Diffusion and Rare Strong Adsorption

An analytic expression is derived to describe the autocorrelation function for fluorophors interacting with heterogeneous chemical interfaces, where both lateral diffusion and reversible strong adsorption occur. The expression is accurate when the rate of strong adsorption is low compared to the rates of both diffusion and desorption, enabling analysis by nonlinear regression. Simulations of single molecules are employed to investigate the applicability of the analytic equation for interpretation of chemical equilibrium and kinetics of single fluorescent molecules undergoing both lateral diffusion and varying amounts of specific adsorption at chemical interfaces. The simulations show that the analytic equation accurately describes the autocorrelation decay, and the equation begins to deviate, as expected, when the adsorption rate becomes large. The results show that a smaller beam size enhances the ability to extract sorption kinetics from the autocorrelation decay, and that a larger beam size enhances the ability to obtain information about the diffusion coefficient with minimal interference from sorption processes. The expression provides a tool for choosing between fluorescence correlation spectroscopy and single-molecule sorting in studies of heterogeneous surfaces.