Highly organized but pliant active site of DNA polymerase β: Compensatory mechanisms in mutant enzymes revealed by dynamics simulations and energy analyses

Linjing Yang, William A. Beard, Samuel H. Wilson, Suse Broyde, Tamar Schlick

Research output: Contribution to journalArticle

Abstract

To link conformational transitions noted for DNA polymerases with kinetic results describing catalytic efficiency and fidelity, we investigate the role of key DNA polymerase β residues on subdomain motion through simulations of five single-residue mutants: Arg-283-Ala, Tyr-271-Ala, Asp-276-Val, Arg-258-Lys, and Arg-258-Ala. Since a movement toward a closed state was only observed for R258A, we suggest that Arg258 is crucial in modulating motion preceding chemistry. Analyses of protein/DNA interactions in the mutant active site indicate distinctive hydrogen bonding and van der Waals patterns arising from compensatory structural adjustments. By comparing closed mutant complexes with the wild-type enzyme, we interpret experimentally derived nucleotide binding affinities in molecular terms: R283A (decreased), Y271A (increased), D276V (increased), and R258A (decreased). Thus, compensatory interactions (e.g., in Y271A with adjacent residues Phe272, Asn279, and Arg 283) increase the overall binding affinity for the incoming nucleotide although direct interactions may decrease. Together with energetic analyses, we predict that R258G might increase the rate of nucleotide insertion and maintain enzyme fidelity as R258A; D276L might increase the nucleotide binding affinity more than D276V; and R283A/K280A might decrease the nucleotide binding affinity and increase misinsertion more than R283A. The combined observations regarding key roles of specific residues (e.g., Arg258) and compensatory interactions echo the dual nature of polymerase active site, namely versatility (to accommodate various basepairs) and specificity (for preserving fidelity) and underscore an organized but pliant active site essential to enzyme function.

Original languageEnglish (US)
Pages (from-to)3392-3408
Number of pages17
JournalBiophysical Journal
Volume86
Issue number6
DOIs
StatePublished - Jun 2004

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DNA-Directed DNA Polymerase
Catalytic Domain
Nucleotides
Enzymes
Hydrogen Bonding
DNA
Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

Highly organized but pliant active site of DNA polymerase β : Compensatory mechanisms in mutant enzymes revealed by dynamics simulations and energy analyses. / Yang, Linjing; Beard, William A.; Wilson, Samuel H.; Broyde, Suse; Schlick, Tamar.

In: Biophysical Journal, Vol. 86, No. 6, 06.2004, p. 3392-3408.

Research output: Contribution to journalArticle

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