title: Direct imaging of single UvrD helicase dynamics on long single-stranded DNA.
abstract: Fluorescence imaging of single-protein dynamics on DNA has been largely limited to double-stranded DNA or short single-stranded DNA. We have developed a hybrid approach for observing single proteins moving on laterally stretched kilobase-sized ssDNA. Here we probed the single-stranded DNA translocase activity of Escherichia coli UvrD by single fluorophore tracking, while monitoring DNA unwinding activity with optical tweezers to capture the entire sequence of protein binding, single-stranded DNA translocation and multiple pathways of unwinding initiation. The results directly demonstrate that the UvrD monomer is a highly processive single-stranded DNA translocase that is stopped by a double-stranded DNA, whereas two monomers are required to unwind DNA to a detectable degree. The single-stranded DNA translocation rate does not depend on the force applied and displays a remarkable homogeneity, whereas the unwinding rate shows significant heterogeneity. These findings demonstrate that UvrD assembly state regulates its DNA helicase activity with functional implications for its stepping mechanism, and also reveal a previously unappreciated complexity in the active species during unwinding.
authors.affiliation: Department of Physics, Center for Physics in Living Cells and Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801-2902, USA.
authors.forename: Kyung Suk
authors.initials: KS
authors.lastname: Lee
authors.forename: Hamza
authors.initials: H
authors.lastname: Balci
authors.forename: Haifeng
authors.initials: H
authors.lastname: Jia
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D057054:Molecular Imaging; D011485:Protein Binding; D021381:Protein Transport
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C506746:UvrD protein, E coli; D004265:DNA Helicases