title: Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore.
abstract: The latch region of the wild-type α-hemolysin (α-HL) protein channel can be used to distinguish single base modifications in double-stranded DNA (dsDNA) via ion channel measurements upon electrophoretic capture of dsDNA in the vestibule of α-HL. Herein, we investigated the use of the latch region to detect a nick in the phosphodiester DNA backbone. The presence of a nick in the phosphodiester backbone of one strand of the duplex results in a significant increase in both the blockade current and noise level relative to the intact duplex. Differentiation between the nicked and intact duplexes based on blockade current or noise, with near baseline resolution, allows real-time monitoring of the rate of T3-DNA ligase-catalyzed phosphodiester bond formation. Under low ionic strength conditions containing divalent cations and a molecular crowding agent (75 mg mL-1PEG), the rate of enzyme-catalyzed reaction in the bulk solution was continuously monitored by electrophoretically capturing reaction substrate or product dsDNA in the α-HL protein channel vestibule. Enzyme kinetic results obtained from the nanopore experiments match those from gel electrophoresis under the same reaction conditions, indicating the α-HL nanopore measurement provides a viable approach for monitoring enzymatic DNA repair activity.
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.
authors.forename: Cherie S
authors.initials: CS
authors.lastname: Tan
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.
authors.forename: Jan
authors.initials: J
authors.lastname: Riedl
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Fleming
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.
authors.forename: Cynthia J
authors.identifier: 0000-0001-7253-8529
authors.initials: CJ
authors.lastname: Burrows
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.
authors.forename: Henry S
authors.identifier: 0000-0002-5053-0996
authors.initials: HS
authors.lastname: White
mesh_terms: D004247:DNA; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D006460:Hemolysin Proteins / Q000378:metabolism*; D007700:Kinetics; D058608:Nanopores
chemical_list: D006460:Hemolysin Proteins; D004247:DNA; D011088:DNA Ligases
keywords: DNA ligase; enzyme kinetics; latch zone; nicked duplex; α-hemolysin