Dynamics of Translocation and Substrate Binding in Individual Complexes Formed with Active Site Mutants of Φ29 DNA Polymerase

Background: Tyr-226 and Tyr-390 in the 29 DNA polymerase active site are implicated in the mechanism of translocation. Results: Y226F and Y390F differ in their effects on translocation and on dNTP and pyrophosphate binding. Conclusion: Mutations in the 29 DNA polymerase and exonuclease active sites perturb dNTP or pyrophosphate binding rates. Significance: DNA polymerase architecture is finely tuned to integrate translocation and substrate binding.The phi 29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual phi 29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the phi 29 DNAP-DNA binary complex and the phi 29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate approximate to 2-fold and increasing the dissociation rate approximate to 10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn2+ rather than Mg2+. The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, approximate to 30 from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb phi 29 DNAP interdomain architecture.