Abstract
During each catalytic cycle, DNA polymerases select deoxyribonucleoside triphosphate (dNTP) substrates complementary to a templating base with high fidelity from a pool that includes noncomplementary dNTPs and both complementary and noncomplementary ribonucleoside triphosphates (rNTPs). The Klenow fragment of Escherichia coli DNA polymerase I (KF) achieves this through a series of conformational transitions that precede the chemical step of phosphodiester bond formation. Kinetic evidence from fluorescence and FRET experiments indicates that discrimination of the base and sugar moieties of the incoming nucleotide occurs in distinct, sequential steps during the selection pathway. Here we show that KF-DNA complexes formed with complementary rNTPs or with noncomplementary nucleotides can be distinguished on the basis of their properties when captured in an electric field atop the α-hemolysin nanopore. The average nanopore dwell time of KF-DNA complexes increased as a function of complementary rNTP concentration. The increase was less than that promoted by complementary dNTP, indicating that the rNTP complexes are more stable than KF-DNA binary complexes but less stable than KF-DNA-dNTP ternary complexes. KF-DNA-rNTP complexes could also be distinguished from KF-DNA-dNTP complexes on the basis of ionic current amplitude. In contrast to complementary rNTPs, noncomplementary dNTPs and rNTPs diminished the average nanopore dwell time of KF-DNA complexes in a concentration-dependent manner, suggesting that binding of a noncomplementary nucleotide keeps the KF-DNA complex in a less stable state. These results imply that nucleotide selection proceeds through a series of complexes of increasing stability in which substrates with the correct moiety promote the forward transitions.
Highlights
To ensure the fidelity of replication, DNA polymerases preferentially incorporate nucleotide substrates complementary to a templating residue and select deoxyribonucleoside triphosphates3 rather than ribonucleoside triphosphates in each catalytic cycle
The single molecule FRET (smFRET) experiments revealed a complex elicited by complementary ribonucleoside triphosphates (rNTPs) that differed in Fluorescence resonance energy transfer (FRET) intensity from the closed complex formed with complementary deoxyribonucleoside triphosphates (dNTPs) [9]
In the smFRET study, the FRET efficiencies of complexes elicited by complementary rNTPs and noncomplementary dNTPs were both consistent with conformations in which the distance between the donor probe in the fingers subdomain and the acceptor probe in the thumb subdomain was intermediate between the fingers-open state that dominates in the KF-DNA binary complex and the fingersclosed state that dominates in complexes formed with complementary dNTPs [9]
Summary
To ensure the fidelity of replication, DNA polymerases preferentially incorporate nucleotide substrates complementary to a templating residue and select deoxyribonucleoside triphosphates (dNTPs) rather than ribonucleoside triphosphates (rNTPs) in each catalytic cycle. The black current trace results from capture of a KF-DNA complex formed with a substrate bearing a block of consecutive abasic (1Ј,2Ј-H) residues (shown in Fig. 2B as red circles) positioned in the DNA template strand such that they reside in the narrow lumen of the nanopore during the EBS and augment the amplitude of this state [13]. Upon this voltage-promoted polymerase dissociation, the duplex DNA segment is drawn into the pore vestibule, which is just wide enough to accommodate it, causing a further current decrease (Fig. 2B, ii, terminal step). Captured DNA molecules that are not bound to KF have the same amplitude and duration of the terminal current step (Fig. 2C, red dots), and these events are quantified
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