Variation in Mutation Rates Caused by RB69pol Fidelity Mutants Can Be Rationalized on the Basis of Their Kinetic Behavior and Crystal Structures

J Mol Biol. 2011 Mar 4;406(4):558-70. doi: 10.1016/j.jmb.2010.12.033. Epub 2011 Jan 7.

Abstract

We have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. However, in vivo results with similar RB69pol nascent base-pair binding pocket mutants showed that mutation rates, as determined by the T4 phage rI forward assay and rII reversion assay, were significantly lower for the RB69pol S565G/Y567A double mutant than for the Y567A single mutant, the opposite of what we would have predicted. To investigate the reasons for this unexpected result, we have determined the pre-steady-state kinetic parameters and crystal structures of relevant ternary complexes. We found that the S565G/Y567A mutant generally had greater base selectivity than the Y567A mutant and that the kinetic parameters for dNMP insertion, excision of the 3'-terminal nucleotide residue, and primer extension beyond a mispair differed not only between these two mutants but also between the two highly mutable sequences in the T4 rI complementary strand. Comparison of the crystal structures of these two mutants with correct and incorrect incoming dNTPs provides insight into the unexpected increase in the fidelity of the S565G/Y567A double mutant. Taken together, the kinetic and structural results provide a basis for integrating and interpreting in vivo and in vitro observations.

Publication types

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

MeSH terms

  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA Mutational Analysis
  • DNA, Viral / metabolism*
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Mutation, Missense
  • Point Mutation
  • Protein Structure, Tertiary
  • Sequence Analysis, DNA
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • DNA, Viral
  • Mutant Proteins
  • Viral Proteins
  • DNA-Directed DNA Polymerase
  • bacteriophage RB69 DNA polymerase

Associated data

  • PIR/3NDK_P
  • PIR/3NDK_T
  • PIR/3NE6_P
  • PIR/3NE6_T
  • PIR/3NGI_P
  • PIR/3NGI_T
  • PIR/3NHG_P
  • PIR/3NHG_T