Single-Molecule Optical-Trapping Techniques to Study Molecular Mechanisms of a Replisome.
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.
['Sun|B|B|', 'Wang|M D|MD|']
D017123:Bacteriophage T7 D004247:DNA D004265:DNA Helicases D004261:DNA Replication D004259:DNA-Directed DNA Polymerase D004926:Escherichia coli D052898:Optical Tweezers D011485:Protein Binding D000072760:Single Molecule Imaging
Replication Helicase Polymerase Slippage Lesion bypass Optical trapping Single molecule