Correct and incorrect nucleotide incorporation pathways in DNA polymerase β

Ravi Radhakrishnan, Tamar Schlick

Research output: Contribution to journalArticle

Abstract

Tracking the structural and energetic changes in the pathways of DNA replication and repair is central to the understanding of these important processes. Here we report favorable mechanisms of the polymerase-catalyzed phosphoryl transfer reactions corresponding to correct and incorrect nucleotide incorporations in the DNA by using a novel protocol involving energy minimizations, dynamics simulations, quasi-harmonic free energy calculations, and mixed quantum mechanics/molecular mechanics dynamics simulations. Though the pathway proposed may not be unique and invites variations, geometric and energetic arguments support the series of transient intermediates in the phosphoryl transfer pathways uncovered here for both the G:C and G:A systems involving a Grotthuss hopping mechanism of proton transfer between water molecules and the three conserved aspartate residues in pol β's active-site. In the G:C system, the rate-limiting step is the initial proton hop with a free energy of activation of at least 17 kcal/mol, which corresponds closely to measured k pol values. Fidelity discrimination in pol β can be explained by a significant loss of stability of the closed ternary complex of the enzyme in the G:A system and much higher activation energy of the initial step of nucleophilic attack, namely deprotonation of terminal DNA primer O3′H group. Thus, subtle differences in the enzyme active-site between matched and mismatched base pairs generate significant differences in catalytic performance.

Original languageEnglish (US)
Pages (from-to)521-529
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume350
Issue number3
DOIs
StatePublished - Nov 24 2006

Fingerprint

DNA-Directed DNA Polymerase
Mechanics
Free energy
Protons
Catalytic Domain
Nucleotides
Humulus
Deprotonation
Molecular mechanics
Proton transfer
DNA Primers
Quantum theory
DNA
Computer simulation
Enzymes
Molecular Dynamics Simulation
DNA Replication
Aspartic Acid
Base Pairing
DNA Repair

Keywords

  • Phosphoryl transfer
  • Polymerase fidelity
  • Quantum mechanics molecular mechanics simulations

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Correct and incorrect nucleotide incorporation pathways in DNA polymerase β. / Radhakrishnan, Ravi; Schlick, Tamar.

In: Biochemical and Biophysical Research Communications, Vol. 350, No. 3, 24.11.2006, p. 521-529.

Research output: Contribution to journalArticle

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