A Mg 2+ ‐induced conformational switch rendering a competent DNA polymerase catalytic complex

The structural and dynamical changes occurring before nucleotide addition were studied using molecular dynamics (MD) simulations of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) complexes containing one or two Mg 2+ ions in the presence of dNTP. Our models revealed that the formation of a catalytically competent DNA polymerase complex required subtle rearrangements at the catalytic site A, which occurred only when an Mg 2+ ion was bound. This model has been validated using pre‐steady‐state kinetics to show that free Mg 2+ is necessary to obtain a catalytically competent polymerase. Kinetic studies carried out with Be 2+ as a cofactor permitted the functional discrimination between metal sites A and B. At low concentrations, Be 2+ increased the catalytic efficiency of the polymerase, while at higher concentrations, it competed with Mg 2+ for binding to site A, and inhibited DNA polymerization. In agreement with experimental data, MD simulations revealed that the catalytic attack distance between the 3′‐OH of the primer and the α phosphorus in complexes containing Be 2+ instead of Mg 2+ at site A was above 4.5 Å. Our findings provide a detailed description of the mechanism of DNA polymerization and should be helpful to understand the molecular basis of DNA replication fidelity. Proteins 2008. © 2007 Wiley‐Liss, Inc.