Multiscale Simulations to Dissect Enzymatic Processing of Nucleic Acids

Nucleic acid polymerization is a key process for genetic inheritance in all living cells. This is performed by a set of DNA/RNA polymerases (Pols) that are effective drug targets in nove therapies. However, DNA damages represent an obstacle for the replication machinery. Here, the role of trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), stand out. Pol-η bypasses ultraviolet-induced basing its function on a highly flexible and conserved R61 and on a transient third ion resolved at the catalytic site in post-reactive state. Nevertheless, how these element assist damaged-DNA replication is still poorly understood. We unravel a highly cooperative mechanism for DNA repair performed by human Pol-η that, via specific R61 conformations, assists the recruitment of the (nucleotide-triphosphate) dNTP in pre-reactive state and ehnances pyrophosphate leaving in post-reactive state. Moreover, bioinformatics analysis revealed that the dNTP always forms an intramolecular H-bond upon formation of the Michaelis-Menten complex. This previously unrecognized H-bond implies a novel self-activated mechanism (SAM), which synergistically connects the in situ nucleophile formation with subsequent nucleotide addition and nucleic acid translocation. Thus, SAM allows an elegant closed-loop sequence of chemical and physical steps for Pols catalysis. Our proposed mechanism is corroborated via ab initio QM/MM simulations on Pol-η. The structural conservation of DNA/RNA Pols supports the extension of SAM to all Pols. Finally, we identified key amino-acids and cations optimally placed nearby the active site of two-metal-ion enzymes and ribozymes such as group-II intron. Such elements interact with the reactants and orient the substrates into the active site, being therefore indispensable for catalysis. Our analysis suggests an unprecedented extension of the two-metal-ion architecture in DNA and RNA polymerases, nucleases and ribozymes. In spite of different biopolymer scaffolds, size and biological function, these enzymes have surprisingly preserved previously-unrecognized positively-charged elements at conserved structural positions to aid DNA and RNA processing.