Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β

Abstract

The I260Q variant of DNA polymerase β is an efficient mutator polymerase with fairly indiscriminate misincorporation activities opposite all template bases. Previous modeling studies have suggested that I260Q harbors structural variations in its hinge region. Here, we present the crystal structures of wild type and I260Q rat polymerase β in the presence and absence of substrates. Both the I260Q apoenzyme structure and the closed ternary complex with double-stranded DNA and ddTTP show ordered water molecules in the hydrophobic hinge near Gln260, whereas this is not the case in the wild type polymerase. Compared to wild type polymerase β ternary complexes, there are subtle movements around residues 260, 272, 295, and 296 in the mutant. The rearrangements in this region, coupled with side chain movements in the immediate neighborhood of the dNTP-binding pocket, namely, residues 258 and 272, provide an explanation for the altered activity and fidelity profiles observed in the I260Q mutator polymerase.

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β

“…47-50) will likely provide mechanistic insights into the how fidelity is governed before closure of the fingers. Our previous work suggests that the hinge region plays a role in substrate discrimination (51)(52)(53) suggesting that the hinge may in some way "sense" the presence of correct versus incorrect dNTP during its initial contact with the fingers domain, and rotate toward a closed complex only if correct dNTP is bound.…”

Cytotoxic Norhopene Triterpenoids from the Bark of Exothea paniculata from Abaco Island, Bahamas

“…Protein-ligand docking studies were carried out based on the crystal structures of yeast topoisomerase II (PDB 1QZR [19] and 2RGR [20]), rat DNA polymerase β (PDB 2BPC [21] and 3 UXN [22]), human 5-lipoxygenase (PDB 3V99 [23]), and human farnesyl protein transferase (PDB 1JCQ [24]). Prior to docking, all solvent molecules and the cocrystallized ligands were removed from the structures.…”

Transitions in DNA polymerase β μs-ms dynamics related to substrate binding and catalysis

“…A large number of X-ray crystallographic structures of pol β with a variety of DNA and dNTP substrates have been determined providing a substantial amount of information on the structure and mechanism of pol β ( 1 , 2 , 5 ). These studies indicate that an elongated form of the enzyme initially re-organizes itself with gapped DNA substrates where the polymerase domain interacts with upstream duplex DNA and the lyase domain interacts with downstream duplex.…”

“… Comparison of X-ray structures of the pol β apoenzyme (3UXN-A ( 5 )), binary complex (3ISB ( 30 )) with 1-nt-gapped DNA, and a ternary complex (2FMS ( 23 )) containing a matched non-hydrolyzable dNTP, dUMPNPP. The structures highlight the transition from fully open apoenzyme to bound and partially closed binary complex to fully closed ternary complex.…”