title: Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase.
abstract: We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,β-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca(2+) and four other structures with catalytically competent Mn(2+) or Mg(2+). These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's α-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα to enable pyrophosphorolysis and assist in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal ion A coordination octahedron. When ptO3' is placed at the vertex of an undistorted, idealized metal ion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 96520-8114, United States.
authors.forename: Shuangluo
authors.initials: S
authors.lastname: Xia
authors.forename: Mina
authors.initials: M
authors.lastname: Wang
authors.forename: Gregor
authors.initials: G
authors.lastname: Blaha
authors.forename: William H
authors.initials: WH
authors.lastname: Konigsberg
authors.forename: Jimin
authors.initials: J
authors.lastname: Wang
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002118:Calcium / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D011756:Diphosphates / Q000737:chemistry; D006860:Hydrogen Bonding; D008274:Magnesium / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D010758:Phosphorus / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics
chemical_list: D011756:Diphosphates; D009692:Nucleic Acid Heteroduplexes; D014764:Viral Proteins; D010758:Phosphorus; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase; D008274:Magnesium; D002118:Calcium