title: "Gate-keeper" residues and active-site rearrangements in DNA polymerase μ help discriminate non-cognate nucleotides.
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.
authors.affiliation: Department of Chemistry and Courant Institute of Mathematical Sciences, New York University, New York, NY, USA.
authors.forename: Yunlang
authors.initials: Y
authors.lastname: Li
authors.forename: Tamar
authors.initials: T
authors.lastname: Schlick
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D020029:Base Pairing; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D056004:Molecular Dynamics Simulation; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055672:Static Electricity
chemical_list: D000596:Amino Acids; D009711:Nucleotides; D004247:DNA; C407743:DNA polymerase mu; D004259:DNA-Directed DNA Polymerase