Mismatch-induced conformational distortions in polymerase beta support an induced-fit mechanism for fidelity

Biochemistry. 2005 Oct 11;44(40):13328-41. doi: 10.1021/bi0507682.

Abstract

Molecular dynamics simulations of DNA polymerase (pol) beta complexed with different incorrect incoming nucleotides (G x G, G x T, and T x T template base x incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol beta structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, alpha-helix N of the thumb subdomain believed to be required for pol beta's catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G x C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (P(alpha)-O3') for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., alpha-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol beta-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Pair Mismatch*
  • Base Pairing
  • Binding Sites
  • Carbon / chemistry
  • Catalysis
  • Computational Biology
  • Crystallography, X-Ray
  • DNA / chemistry
  • DNA Polymerase beta / chemistry*
  • DNA Primers / chemistry
  • DNA-Directed DNA Polymerase / chemistry
  • Humans
  • Ions
  • Kinetics
  • Ligands
  • Magnesium / chemistry
  • Models, Chemical
  • Models, Molecular
  • Molecular Conformation
  • Nucleic Acid Conformation
  • Nucleotides / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Structure-Activity Relationship
  • Time Factors

Substances

  • DNA Primers
  • Ions
  • Ligands
  • Nucleotides
  • Carbon
  • DNA
  • DNA Polymerase beta
  • DNA-Directed DNA Polymerase
  • Magnesium