title: A Mg2+-induced conformational switch rendering a competent DNA polymerase catalytic complex.
abstract: 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 Mg2+ 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 Mg2+ ion was bound. This model has been validated using pre-steady-state kinetics to show that free Mg2+ is necessary to obtain a catalytically competent polymerase. Kinetic studies carried out with Be2+ as a cofactor permitted the functional discrimination between metal sites A and B. At low concentrations, Be2+ increased the catalytic efficiency of the polymerase, while at higher concentrations, it competed with Mg2+ 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 phosphorus in complexes containing Be2+ instead of Mg2+ at site A was above 4.5 A. 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.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Mendieta
authors.forename: Clara E
authors.initials: CE
authors.lastname: Cases-González
authors.forename: Tania
authors.initials: T
authors.lastname: Matamoros
authors.forename: Galo
authors.initials: G
authors.lastname: Ramírez
authors.forename: Luis
authors.initials: L
authors.lastname: Menéndez-Arias
mesh_terms: D001608:Beryllium / Q000737:chemistry / Q000494:pharmacology; D020134:Catalytic Domain; D003198:Computer Simulation; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000187:drug effects*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects
chemical_list: C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D008274:Magnesium; D001608:Beryllium