Green curved arrows show the direction of movement for each labeled residue from the open to closed states. Representative charged residues near the attachment site show variable movement that correspond to the active site residues, and likely continue to move in concert with F762 and Y766 as the enzyme closes and checks the nascent base pair’s electronic structure. The equivalent of KF F762 makes small rotations during each stage of nucleotide incorporation shown, while the equivalent of KF Y766 moves dramatically only from the open to “ajar” state. The open conformation of KF (yellow, PDB ID: 1KFD) can be structurally aligned with the following structures: the homologous Bst Pol I fragment in its open conformation without dNTP bound (light blue, PDB ID: 1元T), its closed conformation (dark blue, PDB ID: 1LV5), and its ajar conformation with a matched base pair (magenta, PDB ID: 3HT3). Model of a KF-functionalized nanocircuit showing various stages of dNTP incorporation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair. 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. 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. 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. All α-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. 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.

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. Each base incorporation accompanied a change in current with its duration defined by τclosed. 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. 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.

Klenow fragment pdf