R964C mutation of DNA polymerase gamma imparts increased stavudine toxicity by decreasing nucleoside analog discrimination and impairing polymerase activity

Abstract

The R964C mutation of human DNA polymerase gamma was recently linked to stavudine (d4T)-mediated mitochondrial toxicity. We utilized pre-steady-state kinetics to determine the effect of this mutation on incorporation of natural substrate dTTP and the active metabolite of d4T (d4TTP). The R964C polymerase gamma holoenzyme demonstrated a 33% decrease in dTTP incorporation efficiency and a threefold-lower d4TTP discrimination relative to that of the wild-type polymerase gamma, providing a mechanistic basis for genetic predisposition to nucleoside reverse transcriptase inhibitor toxicity.

FIG. 1.

(A) Structure of d4T. (B) R964C mutant Pol γ holoenzyme demonstrates a threefold decrease in d4TTP discrimination compared to that of the WT. (C) Molecular model of the Pol γ active site (7). R964 is shown in magenta; O and O1 helices and Y951 are highlighted in yellow. ddCTP is shown bound in the active site (gray) shown with Mg²⁺ ions coordinated with catalytic residues D1135 and E1136 (green). Note the position of the hydroxyl group of Y951, near where the 3′ OH of a natural dNTP would be located. Selected residues mutated in autosomal dominant progressive external ophthalmoplegia are shown in cyan (R943H, Y955C, and A957S) (reviewed in reference 3).