title: Preferred WMSA catalytic mechanism of the nucleotidyl transfer reaction in human DNA polymerase κ elucidates error-free bypass of a bulky DNA lesion.
abstract: Human DNA Pol κ is a polymerase enzyme, specialized for near error-free bypass of certain bulky chemical lesions to DNA that are derived from environmental carcinogens present in tobacco smoke, automobile exhaust and cooked food. By employing ab initio QM/MM-MD (Quantum Mechanics/Molecular Mechanics-Molecular Dynamics) simulations with umbrella sampling, we have determined the entire free energy profile of the nucleotidyl transfer reaction catalyzed by Pol κ and provided detailed mechanistic insights. Our results show that a variant of the Water Mediated and Substrate Assisted (WMSA) mechanism that we previously deduced for Dpo4 and T7 DNA polymerases is preferred for Pol κ as well, suggesting its broad applicability. The hydrogen on the 3'-OH primer terminus is transferred through crystal and solvent waters to the γ-phosphate of the dNTP, followed by the associative nucleotidyl transfer reaction; this is facilitated by a proton transfer from the γ-phosphate to the α,β-bridging oxygen as pyrophosphate leaves, to neutralize the evolving negative charge. MD simulations show that the near error-free incorporation of dCTP opposite the major benzo[a]pyrene-derived dG lesion is compatible with the WMSA mechanism, allowing for an essentially undisturbed pentacovalent phosphorane transition state, and explaining the bypass of this lesion with little mutation by Pol κ.
authors: Department of Chemistry, New York University, NY 10003, USA.
authors: Lee
authors: L
authors: Lior-Hoffmann
authors: Lihua
authors: L
authors: Wang
authors: Shenglong
authors: S
authors: Wang
authors: Nicholas E
authors: NE
authors: Geacintov
authors: Suse
authors: S
authors: Broyde
authors: Yingkai
authors: Y
authors: Zhang
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001580:Benzopyrenes / Q000737:chemistry*; D055162:Biocatalysis; D020134:Catalytic Domain; D018736:DNA Adducts / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011756:Diphosphates / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D010100:Oxygen / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D010753:Phosphoranes / Q000737:chemistry; D011522:Protons; D014867:Water / Q000737:chemistry; D000099270:Y-Family DNA Polymerases
chemical_list: D000596:Amino Acids; D001580:Benzopyrenes; D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D003854:Deoxyribonucleotides; D011756:Diphosphates; D010100:Oxygen; D010710:Phosphates; D010753:Phosphoranes; D011522:Protons; D014867:Water; C478198:benzo(a)pyrene N2-dG adduct; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases