Stabilization of a G-quadruplex from unfolding by replication protein A using potassium and the porphyrin TMPyP4

Abstract

Replication protein A (RPA) plays an essential role in DNA replication by binding and unfolding non-canonical single-stranded DNA (ssDNA) structures. Of the six RPA ssDNA binding domains (labeled A-F), RPA-CDE selectively binds a G-quadruplex forming sequence (5′ -TAGGGGAAGGGTTGGAGTGGGTT- 3′ called Gq23). In K+, Gq23 forms a mixed parallel/antiparallel conformation, and in Na+ Gq23 has a less stable (T M lowered by ∼ 20 ° C), antiparallel conformation. Gq23 is intramolecular and 1D NMR confirms a stable G-quadruplex structure in K+. Full-length RPA and RPA-CDE-core can bind and unfold the Na+ form of Gq23 very efficiently, but complete unfolding is not observed with the K+ form. Studies with G-quadruplex ligands, indicate that TMPyP4 has a thermal stabilization effect on Gq23 in K+, and inhibits complete unfolding by RPA and RPA-CDE-core. Overall these data indicate that G-quadruplexes present a unique problem for RPA to unfold and ligands, such as TMPyP4, could possibly hinder DNA replication by blocking unfolding by RPA. © 2011 Aishwarya Prakash et al.