On careful examination of existing kinetic data for correct and incorrect dNTP incorporations by a variety of DNA polymerases, it is apparent that these enzymes resist a unified description. Instead, the picture that emerges is a rather complex one: for most polymerases, there is evidence for a noncovalent step preceding phosphoryl transfer, but there are less reliable data for determining whether the noncovalent step or phosphoryl transfer is rate-limiting during misincorporation. Although the structural conservation in the polymerase superfamily is probably reflected in a common set of intermediates along the reaction pathway, the energetics of these species vary even when closely related polymerases are compared. Consequently, some polymerases apparently show more discrimination between correctly paired and mispaired dNTPs in the binding step, and polymerases may differ in terms of which step of the reaction is rate-limiting in correct and incorrect insertion reactions. Because of the higher energy barrier in the misincorporation reaction, at least some of the intermediates both before and after the rate-limiting step in the misincorporation pathway will have higher energies than the corresponding intermediates in correct incorporation; consequently, these steps can serve as kinetic checkpoints.Ever since it became possible to measure the fidelity of DNA polymerases, biochemists have sought to understand the source of the amazing specificity of these enzymes, which insert a nucleotide complementary to the templating base with an accuracy far surpassing what would be expected on the basis of the energetics of base pairing (1). Structural studies of DNA polymerases in ternary complexes with a DNA primer-template and the next correct dNTP illustrate the close steric complementarity between the enzyme active site and a Watson-Crick base pair, as well as the presence of hydrogen bonds between the protein and the minor groove side of the nascent base pair (2-6). Both these features could promote fidelity by excluding incorrectly paired dNTPs from the active site. Moreover, the snugly fitting active site could serve to exclude water from the vicinity of the nascent base pair, thus amplifying energetic differences between correct and mispaired nascent base pairs (7). A variety of steps along the reaction pathway could be envisaged as acting as "kinetic checkpoints", serving to test the incoming dNTP for complementarity and facilitating rejection of incorrectly paired dNTPs. One obvious candidate for such a checkpoint is the subdomain movement inferred by comparison of binary (Pol-DNA) and ternary (Pol-DNA-dNTP) complex crystal structures for several polymerases (4,6,(8)(9)(10)(11). This movement of the fingers (dNTP binding) subdomain interconverts the open and closed conformations of the polymerase domain and forms the binding site for the nascent base pair. Structural studies suggest that the Y-family (lesionbypass) DNA polymerases may be an exception. Comparison of unliganded and ternary complex crystal ...

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Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.

The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates.alternating-laser excitation | conformational dynamics | fidelity checkpoints | Klenow fragment | fingers-closing