title: Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.
abstract: Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner.
authors.affiliation: From New England Biolabs, Inc., Ipswich, MA 01938, USA.
authors.forename: Lucia
authors.initials: L
authors.lastname: Greenough
authors.affiliation: From New England Biolabs, Inc., Ipswich, MA 01938, USA.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Schermerhorn
authors.affiliation: From New England Biolabs, Inc., Ipswich, MA 01938, USA.
authors.forename: Laurie
authors.initials: L
authors.lastname: Mazzola
authors.affiliation: From New England Biolabs, Inc., Ipswich, MA 01938, USA.
authors.forename: Joanna
authors.initials: J
authors.lastname: Bybee
authors.affiliation: From New England Biolabs, Inc., Ipswich, MA 01938, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Rivizzigno
authors.affiliation: From New England Biolabs, Inc., Ipswich, MA 01938, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Cantin
authors.affiliation: From New England Biolabs, Inc., Ipswich, MA 01938, USA.
authors.forename: Barton E
authors.initials: BE
authors.lastname: Slatko
authors.affiliation: From New England Biolabs, Inc., Ipswich, MA 01938, USA gardner@neb.com.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Gardner
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics; D053837:DNA Cleavage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics
chemical_list: C022262:Okazaki fragments; D009841:Oligonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D045585:Flap Endonucleases; C468317:FEN1 protein, human; C091139:ribonuclease HII; D016914:Ribonuclease H; D011088:DNA Ligases; D000072481:DNA Ligase ATP