An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces.

Nucleic Acids Research
John G K WilliamsJolene C Egelhoff

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 diss...Continue Reading

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Citations

Jun 26, 2013·Analytical Chemistry·Michael D FurtawJon P Anderson
Sep 10, 2014·Journal of the American Chemical Society·Youngkyu KimJoon Won Park
Nov 10, 2009·Nature Biotechnology·Carl W FullerDmitri V Vezenov
Sep 24, 2010·Chembiochem : a European Journal of Chemical Biology·Ramon Kranaster, Andreas Marx
Oct 11, 2008·Nature Biotechnology·Jeffery A Schloss
Jul 31, 2009·Chemical Communications : Chem Comm·David Loakes, Philipp Holliger
Sep 16, 2017·Nucleic Acids Research·Rubén AgudoLuis Blanco
May 10, 2019·Frontiers in Molecular Biosciences·Andrew F GardnerWilliam E Jack
Jul 28, 2020·Quarterly Reviews of Biophysics·Ali NikoomanzarJohn C Chaput
Aug 20, 2021·ACS Chemical Biology·Adam D CottonIan B Seiple

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Methods Mentioned

BETA
PCR
electrophoresis
affinity purification
infrared

Software Mentioned

Image J
Therminator
Maple

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