title: Base-excision repair activity of uracil-DNA glycosylase monitored using the latch zone of α-hemolysin.
abstract: Nanopores have been investigated as a simple and label-free tool to characterize DNA nucleotides when a ssDNA strand translocates through the constriction of the pore. Here, a wild-type α-hemolysin protein nanopore was used to monitor DNA repair enzyme activity based on base-specific interactions of dsDNA with the vestibule constriction "latch", a previously unrecognized sensing zone in α-hemolysin specific for dsDNA structure. The presence of a single abasic site within dsDNA that is in proximity to the latch zone (±2 nucleotides) results in a large increase in ion channel current, allowing accurate quantitation of the kinetics of base repair reactions involving an abasic site product. Taking advantage of the high resolution for abasic site recognition, the rate of uracil-DNA glycosylase hydrolysis of the N-glycosidic bond, converting 2'-deoxyuridine in DNA to an abasic site, was continuously monitored by electrophoretically capturing reaction substrate or product dsDNA in the ion channel vestibule. Our work suggests use of the nanopore as an enzymology tool and provides a means to identify single base structural changes in dsDNA.
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt City, Utah 84112-0850, United States.
authors.forename: Qian
authors.initials: Q
authors.lastname: Jin
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Fleming
authors.forename: Robert P
authors.initials: RP
authors.lastname: Johnson
authors.forename: Yun
authors.initials: Y
authors.lastname: Ding
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Burrows
authors.forename: Henry S
authors.initials: HS
authors.lastname: White
mesh_terms: D001483:Base Sequence; D004260:DNA Repair; D006460:Hemolysin Proteins / Q000737:chemistry*; D016062:Porosity; D051981:Uracil-DNA Glycosidase / Q000737:chemistry*
chemical_list: D006460:Hemolysin Proteins; D051981:Uracil-DNA Glycosidase