title: DNA translocation of ATP-dependent chromatin remodeling factors revealed by high-resolution optical tweezers.
abstract: ATP-dependent chromatin remodeling complexes (remodelers) use the energy of ATP hydrolysis to regulate chromatin structures by repositioning and reconfiguring nucleosomes. Ensemble experiments have suggested that remodeler ATPases are DNA translocases, molecular motors capable of processively moving along DNA. This concept of DNA translocation has become a foundation for understanding the molecular mechanisms of ATP-dependent chromatin remodeling and its biological functions. However, quantitative characterizations of DNA translocation by representative remodelers are rare. Furthermore, it is unclear how a unified theory of chromatin remodeling is built upon this foundation. To address these problems, high-resolution optical tweezers have been applied to investigate remodeler translocation on bare DNA and nucleosomal DNA substrates at a single-molecule level. Our strategy is to hold two ends of a single DNA molecule and measure remodeler translocation by detecting the end-to-end extension and tension changes of the DNA molecule in response to chromatin remodeling. These single-molecule assays can reveal detailed kinetics of remodeler translocation, including velocity, processivity, stall force, pauses, direction changes, and even step size. Here we describe instruments, reagents, sample preparations, and detailed protocols for the single-molecule experiments. We show that optical tweezer force microscopy is a powerful and friendly tool for studies of chromatin structures and remodeling.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA. yongli.zhang@yale.edu
authors.forename: Yongli
authors.initials: Y
authors.lastname: Zhang
authors.forename: George
authors.initials: G
authors.lastname: Sirinakis
authors.forename: Greg
authors.initials: G
authors.lastname: Gundersen
authors.forename: Zhiqun
authors.initials: Z
authors.lastname: Xi
authors.forename: Ying
authors.initials: Y
authors.lastname: Gao
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D042002:Chromatin Assembly and Disassembly; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D052898:Optical Tweezers / Q000592:standards*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D020080:Tandem Repeat Sequences
chemical_list: D009707:Nucleosomes; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases