title: Use of streptavidin bound to biotinylated DNA structures as model substrates for analysis of nucleoprotein complex disruption by helicases.
abstract: Helicases are a subfamily of translocases that couple the directional translocation along a nucleic acid lattice to the separation of nucleic acid duplexes using the energy derived from nucleoside triphosphate hydrolysis. These enzymes perform essential functions in all aspects of nucleic acid metabolism by unwinding and remodelling DNA or RNA in DNA replication, repair, recombination, transcription and translation. Most classical biochemical studies assay the ability of these enzymes to separate naked nucleic acids. However, many different types of proteins form non-covalent interactions with nucleic acids in vivo and so the true substrates of helicases are protein-nucleic acid complexes rather than naked DNA and RNA. Studies over the last decade have revealed that bound proteins can have substantial inhibitory effects on the ability of helicases to unwind nucleic acids. Any analysis of helicase mechanisms in vitro must therefore consider helicase function within the context of nucleoprotein substrates rather than just DNA or RNA. Here we discuss how to analyse the impact of bound proteins on the ability of helicases to unwind DNA substrates in vitro.
authors.affiliation: Department of Biology, University of York, Wentworth Way, York, YO10 5DD, United Kingdom.
authors.forename: Jan-Gert
authors.initials: JG
authors.lastname: Brüning
authors.affiliation: Department of Biology, University of York, Wentworth Way, York, YO10 5DD, United Kingdom.
authors.forename: Jamieson A L
authors.initials: JA
authors.lastname: Howard
authors.affiliation: Department of Biology, University of York, Wentworth Way, York, YO10 5DD, United Kingdom. Electronic address: peter.mcglynn@york.ac.uk.
authors.forename: Peter
authors.initials: P
authors.lastname: McGlynn
mesh_terms: D001483:Base Sequence / Q000235:genetics; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics*; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D004277:DNA, Single-Stranded; D009698:Nucleoproteins; D004265:DNA Helicases
keywords: DNA unwinding; Motor; Nucleoprotein complex; Protein displacement