Bidendate and tridendate 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-tridendate 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, tridendate 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 tridendate coordination complex. However, complementary dNTPs occasionally form misaligned complexes with mutant RB69 DNA polymerases (RB69pols) that are not in a tridendate coordination state. Here we report finding a β,γ-bidendate 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.

Polymerases:

Topics:

Structure and Structure/Function

Status:

new topics/pols set partial results complete validated

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