Fingers-Closing and Other Rapid Conformational Changes in DNA Polymerase I (Klenow Fragment) and Their Role in Nucleotide Selectivity

Abstract

We have developed a FRET-based assay for the fingers-closing conformational transition that occurs when a binary complex of DNA polymerase I (Klenow fragment) with a primer-template binds a complementary dNTP and have used this and other fluorescence assays to place the fingers-closing step within the reaction pathway. Because the rate of fingers-closing was substantially faster than the rate of nucleotide incorporation measured in chemical quench experiments, fingers-closing cannot be the ratelimiting prechemistry step defined by earlier kinetic studies. Experiments using Ca 2+ instead of Mg 2+ as the metal cofactor suggest instead that the prechemistry step may involve a change in metal ion occupancy at the polymerase active site. The use of ribonucleotide substrates shows there is a base discriminating step that precedes fingers-closing. This earlier step, detected by 2-AP fluorescence, is promoted by complementary nucleotides (ribo-as well as deoxyribo-) but is blocked by mismatches. The complementary rNTP blocks the subsequent fingers-closing step. Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

“…A comparison of the crystal structures of the binary polymerase-DNA complexes with those of the ternary polymerase-DNA-dNTP complexes reveals a substantial movement upon nucleotide binding, supporting the model that fingers closing was the rate-limiting step (7). However, recent results have shown this step is much too fast to be rate limiting, suggesting additional noncovalent steps that must follow it (1,(8)(9)(10)). On the rare occasion of a misinsertion, KF has a 3Ј-5Ј exonuclease proofreading activity that excises the incorrectly base-paired nucleotide.…”

“…during DNA synthesis (1). These studies have shown that most polymerases have similar structural organizations and share a basic mechanism for nucleotide incorporation, although specific differences are evident even between closely related enzymes.…”

“…T he catalytic mechanism of Escherichia coli DNA polymerase I has been rigorously studied for more than 40 years (1,2). The E. coli DNA polymerase I (Klenow fragment [KF]), an active truncated form of polymerase I, is composed of two domains: a polymerase domain that incorporates nucleotides, and a 3Ј-5Ј exonuclease domain that excises misincorporated nucleotides.…”

Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

“…The decrease of the polymerization rate can be explained by T7 DNAp undergoing force-sensitive structural changes during polymerization, when the finger domains have to close to correctly align and incorporate a nucleotide (22). In a physiological setting, our high-tension situation could correspond to an erroneous nucleotide that cannot properly basepair with the template strand, increasing both the probability of unbinding of the incoming nucleotide and the probability of unbinding of DNAp from its pol active site and rebinding with the exo active site (12,23,24). We find that the probability of entering an incorporation pause increases fivefold when the tension is increased to >35 pN.…”

DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase

“…Recent kinetic studies have suggested that a conformational change prior to "finger-closing" may be involved in an early checkpoint for correct dNTP incorporation (7)(8)(9)(10). The conformational changes in the DNAP or the P/T DNA that might be involved in this checkpoint have not been defined.…”