Single-Molecule Analysis and Engineering of DNA Motors

Abstract

Molecular motors are diverse enzymes that transduce chemical energy into mechanical work and, in doing so, perform critical cellular functions such as DNA replication and transcription, DNA supercoiling, intracellular transport, and ATP synthesis. Single-molecule techniques have been extensively used to identify structural intermediates in the reaction cycles of molecular motors and to understand how substeps in energy consumption drive transitions between the intermediates. Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers, atomic force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), nanopore tweezers, and hybrid techniques that increase the number of observables. These methods enable the manipulation of individual biomolecules via the application of forces and torques and the observation of dynamic conformational changes in single motor complexes. We also review how these techniques have been applied to study various motors such as helicases, DNA and RNA polymerases, topoisomerases, nucleosome remodelers, and motors involved in the condensation, segregation, and digestion of DNA. In-depth analysis of mechanochemical coupling in molecular motors has made the development of artificially engineered motors possible. We review techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality. We also discuss how single-molecule analysis of engineered motors allows us to challenge our fundamental understanding of how molecular motors transduce energy.

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules

“…Due to space constraints, we were limited in the number of investigators we could highlight in this section and apologize to those who have contributed to the development and application of single-molecule assays but were not mentioned herein. Specific techniques that utilize TIRF microscopes to study protein-DNA complexes include DNA or protein tethering [39][40][41][42], DNA combing and curtains [43][44][45][46], DNA tightropes (via oblique angle fluorescence imaging) [47,48], as well as smFRET [40,[49][50][51][52][53] and TIRF/AFM systems [54][55][56]. While the examples mentioned below include data collected on both prism and objective-based TIRF instruments, in most cases, the prismTIRF microscope described above can be used for similarly designed experiments.…”

Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules

“…Due to space constraints, we were limited in the number of investigators we could highlight in this section and apologize to those who have contributed to the development and application of single-molecule assays but were not mentioned herein. Specific techniques that utilize TIRF microscopes to study protein-DNA complexes include DNA or protein tethering [36][37][38][39], DNA combing and curtains [40][41][42][43], DNA tightropes (via oblique angle fluorescence imaging) [44,45], as well as smFRET [37,[46][47][48][49][50] and TIRF/AFM systems [51][52][53]. While the examples mentioned below include data collected on both prism and objective based TIRF instruments, in most cases the prismTIRF microscope described above can be used for similarly designed experiments.…”

History of DNA Helicases

“…This high-powered approach, and other innovative strategies in SM experimentation, ushered in a new era for mechanistic studies of DNA helicases. Some of the important and diverse innovations in SM studies of DNA helicases are discussed below from a historical perspective, but due to the broad scope of the field readers are encouraged to consult some timely reviews [207][208][209].…”