The formation and cleavage of phosphodiester bonds is one of the most essential reactions carried out by enzymes. The general mechanistic features of phosphodiester cleavage are universally conserved in ribozymes, polymerases, nucleases, and a wide variety of other nucleic acid processing enzymes such as integrases, transposases or topoisomerases (See ref and references therein). The phosphodiester cleavage generally follows an associative mechanism and utilizes the well-established two metal ion mechanism. Two divalent ions are typically bound by one bridging aspartate coordinating both metal ions, another aspartate and a glutamate (referred to as a DDE motif). Enzymes of the nuclease family are found in all branches of life. Amongst many processes conferred by the nucleases are DNA replication, proofreading, RNA maturation and RNA interference. One of the particularly well-studied nucleases is RNase H. The RNase H fold is conserved in many enzymes with nucleolytic activity including transposases, the argonaute proteins, and the Cas9 enzyme. RNase H is also an essential part of the HIV-1 enzyme machinery as HIV-1 reverse transcriptase (RT) depends on both a polymerase and a RNase H active site localized on two domains for its full functionality. The RNase H subdomain (residues 1021 -1144 of the gag polyprotein, Figure ) is located on the p66 subunit of RT where it catalyzes the removal of the RNA strand in the RNA/DNA hybrid generated by the polymerase. RNase H was found essential for proliferation of HIV-1. In higher eukaryotes RNase H1 is thought to be involved in generating RNA primers for replication and the prevention of genomic instability by removal of RNA nucleotides in R-Loops. Studies of the catalytic mechanism of Bacillus halodurans (Bh) RNase H and human (Hs) RNase H showed the importance of the coordinated movement of two Mg 2+ ions, metal ion preference of Mn and Mg 2+ over Ca 2+ , and possible proton pathways. Recently, Samara and Yang suggested that a third transient Mg ion participates in the catalytic reaction, similarly to polymerases during the reverse reaction. Additionally, Samara and Yang also implicate monovalent ions in the Bh RNase H catalysis. Several key details about the reaction mechanism are still not fully consistent with available data. Kinetic experiments indicate a crucial role of a catalytic histidine in the HIV-1, Hs and E. coli (Ec) RNase H reaction (H1126, H264, H124 in Figure ). All earlier computational studies of the RNase H catalytic reaction were either conducted with the Bh model lacking this crucial histidine or the histidine was not included in the QM region. In similar nucleases with nucleolytic activity, such as CRN-4, TREX1, 3'hExo, or RNase T , a conserved histidine residue (part of the conserved DEDDh motif ) is thought to deprotonate the water nucleophile and for one of these enzymes, namely thesubunit of Ec DNA pol III , QM/MM calculations support this claim. To address several of the outstanding questions regarding the HIV-1 RNase H catalytic mechanism, namely the nature of the general base, the identity of the proton donor of the leaving group, and the role of the third metal ion, we employed classical molecular dynamics (MD) simulations and mixed quantum/classical (QM/MM) simulations utilizing the B3LYP density functional level of theory. We determined the multidimen-sional free energy surface underlying the catalytic reaction of HIV-1 RNase H with two metal ions bound in the active site by combining Hamiltonian replica exchange (RE), umbrella sampling (US), and finite-temperature string method calculations. In addition, we studied the cleavage product resulting from the action of RNase H using classical MD simulations to investigate the role of a third metal ion. Our simulations showed a reaction pathway that differs from those of earlier computational studies but are consistent with kinetic and thiosubstitution experiments for Ec RNase H and Hs RNase H. The main differences to other studies of the RNase H catalytic mechanism are the nature of the general base and the source of the proton for leaving group protonation. Based on our MD simulations we propose that a third divalent cation, necessary for the displacement of the cleaved RNA strand, is recruited from the bulk with the aid of the histidine. In a structural comparison with the catalytic motifs of other nucleases, we are able to show that a conserved histidine plays a dual role in many nucleolytic reactions irrespective of the exact metal ion coordination of the two central Mg 2+ ions in the active site.