title: An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces.
abstract: Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstick surface. Here, we report the genetic modification of the archaeal DNA polymerase 9 degrees N in which two biotinylated peptide 'legs' are inserted at positions flanking the DNA-binding cleft. Streptavidin binding on either side of the cleft both traps the DNA template in the polymerase and orients the complex on a biotinylated surface. We present evidence that purified polymerase-DNA-streptavidin complexes are active both in solution and immobilized on a surface. Processivity is improved from <20 nt in the unmodified polymerase to several thousand nucleotides in the engineered complexes. High-molecular weight DNA synthesized by immobilized complexes is observed moving above the surface even as it remains tethered to the polymerase. Pre-formed polymerase-DNA-streptavidin complexes can be stored frozen and subsequently thawed without dissociation or loss of activity, making them convenient for use in single molecule analysis.
authors: Advanced Research & Development, LI-COR Biosciences, Lincoln, NE, USA. john.williams@licor.com
authors: John G K
authors: JG
authors: Williams
authors: David L
authors: DL
authors: Steffens
authors: Jon P
authors: JP
authors: Anderson
authors: Teresa M
authors: TM
authors: Urlacher
authors: Donald T
authors: DT
authors: Lamb
authors: Daniel L
authors: DL
authors: Grone
authors: Jolene C
authors: JC
authors: Egelhoff
mesh_terms: D019921:Biotinylation; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D005615:Freezing; D007700:Kinetics; D015202:Protein Engineering; D017422:Sequence Analysis, DNA; D019809:Streptavidin / Q000737:chemistry*; D013696:Temperature; D013698:Templates, Genetic
chemical_list: D004247:DNA; D019809:Streptavidin; D004259:DNA-Directed DNA Polymerase