Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

Highlights

• Taq DNA polymerase (Taq Pol) uses conformational changes during catalysis.

• Global transition of all five domains of Taq Pol during dNTP incorporation.

• Conformational dynamics play a critical role in the enzymatic function of Taq Pol.

• Conformational change kinetics of truncated Taq Pol differ from full-length enzyme.

• All DNA polymerases may use global conformational dynamics during polymerization.

Abstract

Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.

Introduction

DNA polymerases are key enzymes in the replication and repair of cellular DNA. Among the six families of DNA polymerases, the A-Family enzymes function in DNA replication, repair and the processing of Okazaki fragments during lagging strand synthesis [1]. Thermus aquaticus DNA polymerase I (Taq Pol) is a thermostable A-Family DNA polymerase that has been the subject of many structural and kinetic investigations due to its widespread laboratory use for DNA amplification in PCR. Taq Pol consists of an N-terminal 5′ → 3′ exonuclease domain and a Klentaq1 domain, analogous to the Klenow fragment of Escherichia coli DNA polymerase I (KF), which contains a polymerase core and an “Intervening” non-functional 3′ → 5′ proofreading exonuclease domain [2], [3]. The polymerase core is further subdivided into Finger, Palm and Thumb domains common to all DNA polymerases. In order to perform the necessary in vivo functions, the functions of the Klentaq1 and N-terminal 5′ → 3′ exonuclease domains of Taq Pol must be tightly coordinated [4], [5], [6].

Early biochemical studies suggested the existence of a rate-limiting non-covalent step prior to the chemistry step for both KF [7], [8], [9] and another A-Family member, T7 DNA polymerase [10], [11]. Subsequently, a large “closing” conformational change in the Finger domain upon dNTP binding was inferred from comparison between binary (Klentaq1·DNA) and ternary (Klentaq1·DNA·dNTP) structures of Klentaq1 [2], [12], [13] and other polymerases including T7 DNA polymerase [14] and human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) [15]. Solution-state stopped-flow fluorescence studies with KF and Klentaq1 provided further evidence for this conformational change and indicated that the conformational change step was much faster than the observed rate-limiting step for correct nucleotide insertion [16], [17], [18], [19]. Contrastingly, in stopped-flow measurements of T7 DNA polymerase, the conformational change step was shown to be less than 2-fold faster than the chemistry step for correct incorporation [20]. Detailed kinetic analysis of the fluorescence data in combination with the data of ³²P-based kinetic assays for both correct and incorrect nucleotide insertions by T7 DNA polymerase suggested a mechanism by which the rate of the reverse conformational change plays a critical role in determining substrate specificity [20], [21]. In these studies, it was argued that the Finger domain closing upon binding to a correct nucleotide leads to a tight ternary complex committing the substrate to catalysis, while an incorrect nucleotide leads to a misformed ternary complex that favors substrate dissociation over nucleotide incorporation. Similarly, in an investigation with HIV-1 RT, it was shown that nucleotide binding was a two-step process involving a conformational change and that rate of the reverse conformational change step relative to the rate of the subsequent chemistry step was a key factor influencing the selective incorporation of a dCTP over a nucleoside analog drug [22]. Further investigations have provided evidence for a multi-step nucleotide binding mechanism involving additional rapid conformational changes occurring before Finger domain closing in Klentaq1 and Klenow that may also aid in the selection of correct nucleotides [16], [23], though the structural nature of these steps is not entirely clear. Interestingly, single-molecule evidence has suggested that both open and closed conformations may be sampled by a DNA polymerase even in the absence of DNA and/or nucleotide substrates and that the presence of nucleotide shifts the conformational equilibrium toward the closed state [24], [25], [26], [27]. Furthermore, crystallographic [28] and single-molecule [25] investigations have demonstrated the existence of a third conformational state of the Finger domain distinct from the open and closed states, which is stabilized in the presence of a mismatched nucleotide. In a recent study, high-precision Förster resonance energy transfer (FRET) measurements demonstrated how the presence of matched and mismatched nucleotides can alter the conformational equilibrium between these three Finger domain conformations for Klentaq1 [27], underscoring the importance of this conformational change as a nucleotide complementarity selection mechanism.

Although there has been extensive study of the closing motion of the Finger domain, structural studies have revealed little to no conformational change beyond the Finger domain [2], [12], [13], and thus little has been performed to investigate the solution-state dynamics of the other domains of the A-Family DNA polymerases. However, while structural studies of the Y-Family DNA polymerases have revealed no obvious conformational change analogous to the Finger domain closing in other DNA polymerases, our recent studies with Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-Family DNA polymerase, have revealed global conformational changes in all four domains of the enzyme [29], [30]. Additionally, molecular dynamics simulations have shown that short-distance conformational changes throughout the structure of HIV-1 RT accompany the larger Finger domain closing during nucleotide binding [31]. Previous studies on the conformational dynamics of Klentaq1 did not address the effects of the presence of the N-terminal domain in the full-length Taq Pol. However, neutron spin echo experiments have indicated that the conformational dynamics of the Klentaq1 domain and the N-terminal domain are strongly coupled [32]. In addition, biochemical studies have demonstrated that the kinetics of both exonuclease and polymerization activities differ for full-length and truncated forms of the enzyme [33], [34], [35], [36]. Therefore, in this study, we developed a FRET system to independently monitor the conformational dynamics of all five domains of full-length Taq Pol relative to the DNA substrate during the binding and incorporation of correct dNTPs. Furthermore, we introduced a de novo disulfide bond into Taq Pol to limit the conformational flexibility of the Finger and Thumb domains in order to gain insight into the contribution of this motion to the nucleotide incorporation efficiency of Taq Pol.