Recognition and Binding of Human Telomeric G-Quadruplex DNA by Unfolding Protein 1

Abstract

The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG) _n . UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG) ₃] G-quadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1–G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na ⁺ and K ⁺ G-quadruplex–UP1 complexes (Δ H values of −43 and −19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na ⁺ form of G-quadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 10 ⁸ M ^–1 (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 10 ⁶ M ^–1 (strand)]. Circular dichroism spectroscopy reveals the Na ⁺ form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure.