title: Single-Molecule Optical-Trapping Techniques to Study Molecular Mechanisms of a Replisome.
abstract: The replisome is a multiprotein molecular machinery responsible for the replication of DNA. It is composed of several specialized proteins each with dedicated enzymatic activities, and in particular, helicase unwinds double-stranded DNA and DNA polymerase catalyzes the synthesis of DNA. Understanding how a replisome functions in the process of DNA replication requires methods to dissect the mechanisms of individual proteins and of multiproteins acting in concert. Single-molecule optical-trapping techniques have proved to be a powerful approach, offering the unique ability to observe and manipulate biomolecules at the single-molecule level and providing insights into the mechanisms of molecular motors and their interactions and coordination in a complex. Here, we describe a practical guide to applying these techniques to study the dynamics of individual proteins in the bacteriophage T7 replisome, as well as the coordination among them. We also summarize major findings from these studies, including nucleotide-specific helicase slippage and new lesion bypass pathway in T7 replication.
authors.affiliation: School of Life Science and Technology, ShanghaiTech University, Shanghai, PR China.
authors.forename: B
authors.initials: B
authors.lastname: Sun
authors.affiliation: Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, NY, United States; Howard Hughes Medical Institute, Cornell University, Ithaca, NY, United States. Electronic address: mwang@physics.cornell.edu.
authors.forename: M D
authors.initials: MD
authors.lastname: Wang
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry; D004261:DNA Replication / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D052898:Optical Tweezers; D011485:Protein Binding; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases
keywords: Helicase; Lesion bypass; Optical trapping; Polymerase; Replication; Single molecule; Slippage