Structures of Ternary Complexes of Rat DNA Polymerase β, a DNA Template-Primer, and ddCTP

TLDR

ddCTP, the triphosphate of the nucleoside analog dideoxycytidine used to treat AIDS, and conserved Asp residues (Asp190 and Asp256) are common to all studied polymerases. The study aims to describe a two‑metal‑ion mechanism for nucleotidyl transfer that may apply to all polymerases. In the pol β active site, the primer 3′‑OH, ddCTP phosphates, and two Mg²⁺ ions cluster around Asp190, Asp192, and Asp256, supporting the two‑metal‑ion mechanism. Crystal structures of two rat pol β ternary complexes with ddCTP at 2.9 and 3.6 Å reveal similar binding despite different space groups and a distinct DNA template‑primer interaction compared to other polymerase‑DNA models.

Abstract

Two ternary complexes of rat DNA polymerase β (pol β), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 Å and 3.6 Å resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol β active site, the attacking 3′-OH of the elongating primer, the ddCTP phosphates, and two Mg 2+ ions are all clustered around Asp 190 , Asp 192 , and Asp 256 . Two of these residues, Asp 190 and Asp 256 , are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases—the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol β—whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol β binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.