The Critical Role of Magnesium Ions in DNA Polymerase 's Closing and Active Site Assembly

To dissect the effects of the nucleotide-binding and catalytic metal ions on DNA polymerase mechanisms for DNA repair and synthesis, aside from the chemical reaction, we investigate their roles in the conformational transitions between closed and open states and assembly/disassembly of the active site of a pol beta/DNA complex before and after the chemical step of nucleotide incorporation. Using dynamics simulations, we find that closing before chemistry requires both divalent metal ions in the active site while opening after chemistry is triggered by release of the catalytic metal ion. The critical closing before chemistry is stabilized by the interaction of the incoming nucleotide with conserved catalytic residues (Asp190, Asp192, Asp256) and the two functional magnesium ions; without the catalytic ion, other protein residues (Arg180, Arg183, Gly189) coordinate the incomer's triphosphate group through the nucleotide-binding ion. Because we also note microionic heterogeneity near the active site -- Mg2+ and Na+ ions can diffuse into the active site relatively rapidly -- we suggest that the binding of the catalytic Mg2+ itself is not a rate-limiting conformational or overall step. However, geometric adjustments associated with ion and proper positioning in the active site, including subtle but systematic motions of protein side chains (e.g., Asp192, Arg258), define slow or rate-limiting conformational steps that may guide fidelity mechanisms. These sequential rearrangements are likely sensitively affected when an incorrect nucleotide approaches the active site. Our suggestion -- that subtle and slow adjustments of the nucleotide-binding and catalytic magnesium ions help guide polymerase selection for the correct nucleotide -- extends descriptions of polymerase pathways and underscores the importance of the delicate conformational events both before and after the chemical reaction to polymerase efficiency and fidelity mechanisms.

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