title: Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits.
abstract: DNA polymerases exhibit a surprising tolerance for analogs of deoxyribonucleoside triphosphates (dNTPs), despite the enzymes' highly evolved mechanisms for the specific recognition and discrimination of native dNTPs. Here, individual DNA polymerase I Klenow fragment (KF) molecules were tethered to a single-walled carbon nanotube field-effect transistor (SWCNT-FET) to investigate accommodation of dNTP analogs with single-molecule resolution. Each base incorporation accompanied a change in current with its duration defined by τclosed. Under Vmax conditions, the average time of τclosed was similar for all analog and native dNTPs (0.2 to 0.4 ms), indicating no kinetic impact on this step due to analog structure. Accordingly, the average rates of dNTP analog incorporation were largely determined by durations with no change in current defined by τopen, which includes molecular recognition of the incoming dNTP. All α-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. During polymerization with 6-Cl-2APTP, 2-thio-dTTP, or 2-thio-dCTP, the nanocircuit uncovered an alternative conformation represented by positive current excursions that does not occur with native dNTPs. A model consistent with these results invokes rotations by the enzyme's O-helix; this motion can test the stability of nascent base pairs using nonhydrophilic interactions and is allosterically coupled to charged residues near the site of SWCNT attachment. This model with two opposing O-helix motions differs from the previous report in which all current excursions were solely attributed to global enzyme closure and covalent-bond formation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair.
authors.affiliation: Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
authors.forename: Kaitlin M
authors.initials: KM
authors.lastname: Pugliese
authors.affiliation: Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
authors.forename: O Tolga
authors.initials: OT
authors.lastname: Gul
authors.affiliation: Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
authors.forename: Yongki
authors.initials: Y
authors.lastname: Choi
authors.affiliation: Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
authors.forename: Tivoli J
authors.initials: TJ
authors.lastname: Olsen
authors.affiliation: Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Sims
authors.affiliation: Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Collins
authors.affiliation: Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Weiss
mesh_terms: D004256:DNA Polymerase I / Q000737:chemistry* / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D037742:Nanotubes, Carbon / Q000737:chemistry*; D011122:Polyphosphates / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003854:Deoxyribonucleotides; D037742:Nanotubes, Carbon; D011122:Polyphosphates; D004256:DNA Polymerase I; C005692:triphosphoric acid