title: Bidentate and tridentate metal-ion coordination states within ternary complexes of RB69 DNA polymerase.
abstract: Two divalent metal ions are required for primer-extension catalyzed by DNA polymerases. One metal ion brings the 3'-hydroxyl of the primer terminus and the α-phosphorus atom of incoming dNTP together for bond formation so that the catalytically relevant conformation of the triphosphate tail of the dNTP is in an α,β,γ-tridentate coordination complex with the second metal ion required for proper substrate alignment. A probable base selectivity mechanism derived from structural studies on Dpo4 suggests that the inability of mispaired dNTPs to form a substrate-aligned, tridentate coordination complex could effectively cause the mispaired dNTPs to be rejected before catalysis. Nevertheless, we found that mispaired dNTPs can actually form a properly aligned tridentate coordination complex. However, complementary dNTPs occasionally form misaligned complexes with mutant RB69 DNA polymerases (RB69pols) that are not in a tridentate coordination state. Here, we report finding a β,γ-bidentate coordination complex that contained the complementary dUpNpp opposite dA in the structure of a ternary complex formed by the wild type RB69pol at 1.88 Å resolution. Our observations suggest that several distinct metal-ion coordination states can exist at the ground state in the polymerase active site and that base selectivity is unlikely to be based on metal-ion coordination alone.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
authors.forename: Shuangluo
authors.initials: S
authors.lastname: Xia
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Eom
authors.forename: William H
authors.initials: WH
authors.lastname: Konigsberg
authors.forename: Jimin
authors.initials: J
authors.lastname: Wang
mesh_terms: D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D011487:Protein Conformation; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D002413:Cations, Divalent; D003854:Deoxyribonucleotides; D008670:Metals; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase; D008274:Magnesium; D002118:Calcium