The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg 2؉ ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp 705 and Asp 882 , of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp 882 ; this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp 882 position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp 705 carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg 2؉ into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg 2؉ concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing.DNA polymerases catalyze phosphodiester bond formation between the 3Ј-OH of a DNA primer strand and the ␣-phosphorus of an incoming deoxynucleoside triphosphate. Before covalent bond formation, the enzyme-substrate complex undergoes a series of noncovalent changes that prepare the active site for the chemical step and also serve as kinetic checkpoints to ensure selection of the correct nucleotide (1). In the Klenow fragment of DNA polymerase I (Pol I(KF)) 2 , the subject of the present study, there is evidence for at least three prechemistry steps following the binding of nucleotide, as shown in the reaction pathway of Fig. 1.Two prechemistry steps in Pol I(KF) have been observed directly by the use of fluorescent probes. The first of these conformational transitions (step 2.1) is a DNA rearrangement, detected by a 2-aminopurine (2-AP) reporter placed 5Ј to the templating base (2). The following step (step 2.2), detected using a FRET-based assay (3), is the closing of the fingers subdomain, which had been inferred from the comparison of DNA polymerase binary (Pol-DNA) and ternary (Pol-DNA-dNTP) cocrystal structures ( Fig. 2) (4 -8). The order of these two conformational transitions in the reaction pathway was deduced from the behavior of ribonucleotide substrates. With an incoming rNTP complementary to the templating base, the DNA rearrangement takes place normally, but fingers-closing is stro...

Background: mtSSB stimulates the activity of Pol ␥. Results: Stimulation of Pol ␥ activity by SSB correlates with the organization of ssDNA templates in a species-independent manner. Conclusion: Organization of the template DNA by mtSSB is the major factor contributing to the stimulation of Pol ␥ activity. Significance: This study provides insight into the functional relationship of Pol ␥ and mtSSB and a general mechanism for it.

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