Summary information and primary citation

PDB-id
4ds5; DSSR-derived features in text and JSON formats
Class
transferase-DNA
Method
X-ray (1.68 Å)
Summary
Ternary complex of bacillus DNA polymerase i large fragment, DNA duplex, and rctp in presence of mg2+
Reference
Wang W, Wu EY, Hellinga HW, Beese LS (2012): "Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides." J.Biol.Chem., 287, 28215-28226. doi: 10.1074/jbc.M112.366609.
Abstract
In addition to discriminating against base-pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxy nucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2' hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present ten high-resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment (BF) variants complexed with deoxy-, ribo-, dideoxy-nucleotides, and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites, and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base-pair discrimination.

Cartoon-block schematics in six views (download the tarball)

PyMOL session file Download PDB file View in 3Dmol.js