Simultaneous Mechanical and Fluorescence Detection of Helicase-Catalyzed DNA Unwinding.

ABSTRACT

Helicases are ubiquitous molecular motor proteins that utilize the energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to transiently convert the duplex form of nucleic acids to single-stranded intermediates for many biological processes. These enzymes play vital roles in nearly all aspects of nucleic acid metabolism, such as DNA repair and RNA splicing. Understanding helicase's functional roles requires methods to dissect the mechanisms of motor proteins at the molecular level. In the past three decades, there has been a large increase in the application of single-molecule approaches to investigate helicases. These techniques, such as optical tweezers and single-molecule fluorescence, offer capabilities to monitor helicase motions with unprecedented spatiotemporal resolution, to apply quantitative forces to probe the chemo-mechanical activities of these motors and to resolve helicase heterogeneity at the single-molecule level. In this chapter, we describe a single-molecule method that combines optical tweezers with confocal fluorescence microscopy to study helicase-catalyzed DNA unwinding. Using Bloom syndrome protein (BLM), a multifunctional helicase that maintains genome stability, as an example, we show that this method allows for the simultaneous detection of displacement, force and fluorescence signals of a single DNA molecule during unwinding in real time, leading to the discovery of a distinct bidirectional unwinding mode of BLM that is activated by a single-stranded DNA binding protein called replication protein A (RPA). We provide detailed instructions on how to prepare two DNA templates to be used in the assays, purify the BLM and RPA proteins, perform single-molecule experiments, and acquire and analyse the data.