Stabilization of a G-Quadruplex from Unfolding by Replication Protein A Using Potassium and the Porphyrin TMPyP4

Abstract EN

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 (TM 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.