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Sequential Side-Chain Residue Motions Transform the Binary into the Ternary State of DNA Polymerase

Department of Chemistry and Courant Institute of Mathematical Sciences, New York University, New York, New York

Correspondence: Address reprint requests to Tamar Schlick, Tel.: 212-998-3116; E-mail: schlick{at}nyu.edu.

The nature of conformational transitions in DNA polymerase (pol ), a low-fidelity DNA repair enzyme in the X-family that fills short nucleotide gaps, is investigated. Specifically, to determine whether pol has an induced-fit mechanism and open-to-closed transition before chemistry, we analyze a series of molecular dynamics simulations from both the binary and ternary states before chemistry, with and without the incoming nucleotide, with and without the catalytic Mg²⁺ ion in the active site, and with alterations in active site residues Ile⁴⁹² and Arg⁵¹⁷. Though flips occurred for several side-chain residues (Ile⁴⁹², Tyr⁵⁰⁵, Phe⁵⁰⁶) in the active site toward the binary (inactive) conformation and partial DNA motion toward the binary position occurred without the incoming nucleotide, large-scale subdomain motions were not observed in any trajectory from the ternary complex regardless of the presence of the catalytic ion. Simulations from the binary state with incoming nucleotide exhibit more thumb subdomain motion, particularly in the loop containing ß-strand 8 in the thumb, but closing occurred only in the Ile⁴⁹²Ala mutant trajectory started from the binary state with incoming nucleotide and both ions. Further connections between active site residues and the DNA position are also revealed through our Ile⁴⁹²Ala and Arg⁵¹⁷Ala mutant studies. Our combined studies suggest that while pol does not demonstrate large-scale subdomain movements as DNA polymerase ß (pol ß), significant DNA motion exists, and there are sequential subtle side chain and other motions—associated with Arg⁵¹⁴, Arg⁵¹⁷, Ile⁴⁹², Phe⁵⁰⁶, Tyr⁵⁰⁵, the DNA, and again Arg⁵¹⁴ and Arg⁵¹⁷—all coupled to active site divalent ions and the DNA motion. Collectively, these motions transform pol to the chemistry-competent state. Significantly, analogs of these residues in pol ß (Lys²⁸⁰, Arg²⁸³, Arg²⁵⁸, Phe²⁷², and Tyr²⁷¹, respectively) have demonstrated roles in determining enzyme efficiency and fidelity. As proposed for pol ß, motions of these residues may serve as gate-keepers by controlling the evolution of the reaction pathway before the chemical reaction.