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J. Biol. Chem., Vol. 278, Issue 7, 5072-5081, February 14, 2003

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Rapid Segmental and Subdomain Motions of DNA Polymerase ^*

Soon-Jong Kim^§, William A. Beard, John Harvey^¶, David D. Shock, Jay R. Knutson^¶, and Samuel H. Wilson

From the  Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the ^¶ Laboratory of Biophysical Chemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

DNA polymerase (pol)  is a two-domain DNA repair enzyme that undergoes structural transitions upon binding substrates. Crystallographic structures indicate that these transitions include movement of the amino-terminal 8-kDa lyase domain relative to the 31-kDa polymerase domain. Additionally, a polymerase subdomain moves toward the nucleotide-binding pocket after nucleotide binding, resulting in critical contacts between -helix N and the nascent base pair. Kinetic and structural characterization of pol  has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the microenvironment and dynamics of both the lyase domain and -helix N in the polymerase domain, the single native tryptophan (Trp-325) of wild-type enzyme was replaced with alanine, and tryptophan was strategically substituted for residues in the lyase domain (F25W/W325A) or near the end of -helix N (L287W/W325A). Influences of substrate on the fluorescence anisotropy decay of these single tryptophan forms of pol  were determined. The results revealed that the segmental motion of -helix N was rapid (~1 ns) and far more rapid than the step that limits chemistry. Binding of Mg²⁺ and/or gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in -helix N. More important, binding of a correct nucleotide significantly limited the angular range of the nanosecond motion within -helix N. In contrast, the segmental motion of the 8-kDa domain was "frozen" upon DNA binding alone, and this restriction did not increase further upon nucleotide binding. The dynamics of -helix N are discussed from the perspective of the "open" to "closed" conformational change of pol  deduced from crystallography, and the results are more generally discussed in the context of reaction cycle-regulated flexibility for proteins acting as molecular motors.

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