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

¹ New York University
² The Scripps Research Institute

^* To whom correspondence should be addressed. E-mail: schlick{at}nyu.edu.

Submitted on June 23, 2006
Revised on July 20, 2006
Accepted on 1 August 2006

	Abstract

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 towards 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 -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 (pol ), 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 (Lys280, Arg283, Arg258, Phe272, and Tyr271, respectively) have demonstrated roles in determining enzyme efficiency and fidelity. As proposed for pol , motions of these residues may serve as gate-keepers by controlling the evolution of the reaction pathway before the chemical reaction.

Key Words: Conformational Changes, Induced-fit mechanism, Molecular Dynamics simulations, Protein/DNA complex, pol , polymerase