RESUMEN
DNA polymerases maintain genomic integrity by copying DNA with high fidelity, part of which relies on the polymerase fingers opening-closing transition, a series of conformational changes during the DNA synthesis reaction cycle. Fingers opening and closing has been challenging to study, mainly due to the need to synchronise molecular ensembles. We previously studied fingers opening-closing on single polymerase-DNA complexes using single-molecule FRET; however, our work was limited to pre-chemistry reaction steps. Here, we advance our analysis to extensible substrates, and observe DNA polymerase (Pol) conformational changes across the entire DNA polymerisation reaction in real-time, gaining direct access to an elusive post-chemistry step rate-limiting for DNA synthesis. Our results showed that Pol adopts the fingers-closed conformation during polymerisation, and that the post-chemistry rate-limiting step occurs in the fingers-closed conformation. We found that fingers-opening in the Pol-DNA binary complex in the absence of polymerisation is slow (â¼5.3 s-1), and comparable to the rate of fingers-opening after polymerisation (3.4 s-1); this indicates that the fingers-opening step itself could be largely responsible for the slow post-chemistry step, with the residual rate potentially accounted for by pyrophosphase release. We also observed that DNA chain-termination of the 3' end of the primer increases substantially the rate of fingers-opening in the Pol-DNA binary complex (5.3 â 29 s-1), demonstrating that the 3'-OH residue is important for the kinetics of fingers conformational changes. Our observations offer mechanistic insight and tools to offer mechanistic insight for all nucleic acid polymerases.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Cristalografía por Rayos X , Escherichia coli , Cinética , Modelos Moleculares , Nucleótidos , Conformación ProteicaRESUMEN
DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN/biosíntesis , ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleótidos/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Movimiento (Física) , Conformación ProteicaRESUMEN
We have previously introduced tethered fluorophore motion (TFM), a single-molecule fluorescence technique that monitors the effective length of a biopolymer such as DNA. TFM uses the same principles as tethered particle motion (TPM) but employs a single fluorophore in place of the bead, allowing TFM to be combined with existing fluorescence techniques on a standard fluorescence microscope. TFM has been previously been used to reveal the mechanism of two site-specific recombinase systems, Cre-loxP and XerCD-dif. In this work, we characterize TFM, focusing on the theoretical basis and potential applications of the technique. Since TFM is limited in observation time and photon count by photobleaching, we present a description of the sources of noise in TFM. Comparing this with Monte Carlo simulations and experimental data, we show that length changes of 100 bp of double-stranded DNA are readily distinguishable using TFM, making it comparable with TPM. We also show that the commonly recommended pixel size for single-molecule fluorescence approximately optimizes signal to noise for TFM experiments, thus enabling facile combination of TFM with other fluorescence techniques, such as Förster resonance energy transfer (FRET). Finally, we apply TFM to determine the polymerization rate of the Klenow fragment of DNA polymerase I, and we demonstrate its combination with FRET to observe synapsis formation by Cre using excitation by a single laser. We hope that TFM will be a useful addition to the single-molecule toolkit, providing excellent insight into protein-nucleic acid interactions.