"Gate-keeper" residues and active-site rearrangements in DNA polymerase μ help discriminate non-cognate nucleotides

PLoS Comput Biol. 2013;9(5):e1003074. doi: 10.1371/journal.pcbi.1003074. Epub 2013 May 23.

Abstract

Incorporating the cognate instead of non-cognate substrates is crucial for DNA polymerase function. Here we analyze molecular dynamics simulations of DNA polymerase μ (pol μ) bound to different non-cognate incoming nucleotides including A:dCTP, A:dGTP, A(syn):dGTP, A:dATP, A(syn):dATP, T:dCTP, and T:dGTP to study the structure-function relationships involved with aberrant base pairs in the conformational pathway; while a pol μ complex with the A:dTTP base pair is available, no solved non-cognate structures are available. We observe distinct differences of the non-cognate systems compared to the cognate system. Specifically, the motions of active-site residue His329 and Asp330 distort the active site, and Trp436, Gln440, Glu443 and Arg444 tend to tighten the nucleotide-binding pocket when non-cognate nucleotides are bound; the latter effect may further lead to an altered electrostatic potential within the active site. That most of these "gate-keeper" residues are located farther apart from the upstream primer in pol μ, compared to other X family members, also suggests an interesting relation to pol μ's ability to incorporate nucleotides when the upstream primer is not paired. By examining the correlated motions within pol μ complexes, we also observe different patterns of correlations between non-cognate systems and the cognate system, especially decreased interactions between the incoming nucleotides and the nucleotide-binding pocket. Altered correlated motions in non-cognate systems agree with our recently proposed hybrid conformational selection/induced-fit models. Taken together, our studies propose the following order for difficulty of non-cognate system insertions by pol μ: T:dGTP<A(syn):dATP<T:dCTP<A:dGTP<A(syn):dGTP<A:dCTP<A:dATP. This sequence agrees with available kinetic data for non-cognate nucleotide insertions, with the exception of A:dGTP, which may be more sensitive to the template sequence. The structures and conformational aspects predicted here are experimentally testable.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acids / chemistry*
  • Amino Acids / metabolism
  • Amino Acids / physiology
  • Base Pairing
  • Catalytic Domain*
  • DNA / chemistry
  • DNA / metabolism
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism
  • DNA-Directed DNA Polymerase / physiology
  • Molecular Dynamics Simulation
  • Nucleotides / chemistry*
  • Nucleotides / metabolism
  • Protein Binding
  • Protein Conformation
  • Static Electricity

Substances

  • Amino Acids
  • Nucleotides
  • DNA
  • DNA polymerase mu
  • DNA-Directed DNA Polymerase

Grants and funding

This work was supported in part by Philip Morris USA Inc., Philip Morris International, and by NSF award MCB-0316771. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.