Structural Insights into Complete Metal Ion Coordination from Ternary Complexes of B Family RB69 DNA Polymerase
- Yale
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'-{alpha},{beta}-substituted triphosphate, dUpXpp, where X is either NH or CH{sub 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{sup 2+} and four other structures with catalytically competent Mn{sup 2+} or Mg{sup 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 {alpha}-phosphorus atom (P{alpha}) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's {alpha}-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach P{alpha}. 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 P{alpha} 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 P{alpha}. This geometric relationship appears to be conserved among DNA polymerases of known structure.
- Research Organization:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Organization:
- FOREIGNNIH
- OSTI ID:
- 1028007
- Journal Information:
- Biochemistry (Eaton), Vol. 50, Issue 42; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- ENGLISH
Similar Records
Using a Fluorescent Cytosine Analogue tC[superscript o] To Probe the Effect of the Y567 to Ala Substitution on the Preinsertion Steps of dNMP Incorporation by RB69 DNA Polymerase
Substitution of Ala for Tyr567 in RB69 DNA Polymerase Allows dAMP and dGMP To Be Inserted opposite Guanidinohydantoin