We also identified a highly conserved glutamate residue as the most likely proton donor for the leaving group phosphate O3', providing evidence why a glutamate and not a fourth aspartate is present in the DEDD motif in RNase H. Furthermore, to shed light on the post-reaction product release process, we performed MD simulations to identify the role of active-site metal ions and the conserved histidine residue. We sampled the possible metal ion binding sites of the cleaved product state for transiently bound mono-and divalent cations in multiple, 100 nslong MD simulations. Our simulations show that a third metal cation is recruited from the bulk finding two potential cation binding sites that are consistent with those identified experimentally by Samara and Yang 10 and Gan et. al. . Our results suggest that the third MgC binding site plays a key role in the product release based on the cleaved product stability in our MD simulations, aided also by the conserved histidine, which has not been reported before. Our results confirm that this mechanism is likely more general for other nucleases, based on structural similarity with Bh RNase H, HIV-1 RNase H and Aa-RNase II crystallographic data. Through the novel understanding of structural and mechanistic roles of key conserved residues, we are able to generalize the nucleic acid cleavage reaction mechanism to a wide array of biomedically very important nucleases, aiding structural and functional understanding into this ubiquitous biochemical process.