Interaction of Replication Protein-A with G-Quadruplex Structures

Abstract

We performed systematic single molecule and bulk studies on the interactions of Replication Protein A (RPA) with four different G-Quadruplex (GQ) structures. RPA, the most abundant single strand DNA binding protein in eukaryotes, is known to interact and unfold GQ structures however; this unfolding activity has not been systematically studied for different GQ structures. As a first step to study this dependence, we studied RPA-GQ interactions for GQ structures in which we varied the number of tetrad layers while keeping the loop length and loop sequence fixed. We studied GQ forming sequences of the form TT (G_n TTT)_3 G_n TT where 1< n <6. These sequences correspond to 2-5 layer GQ constructs (L2-L5) with 3 nucleotide loops (TTT) and two nucleotide overhang sequences (TT). Our results showed that 100%, 80%, 50%, and 20% of the L2, L3, L4, and L5 GQ molecules were unfolded in equilibrium and physiological RPA concentration. The systematic, almost linear, increase in stability of GQ structure as the number of layers is increased is very interesting and will provide an important ingredient in understanding protein-GQ interactions. In addition, we measured the timescales of GQ folding as a result of K+ addition, and GQ unfolding by RPA using a single molecule stopped flow type measurement. We found that the folding of all GQ constructs takes a similar time of 0.3±0.1 sec, with the exception of L2. L2 has two distinct transitions with transition times of 0.3±0.1 sec and 0.5±0.1 sec, and the longer transition time demonstrates a clear intermediate folding conformation. Surprisingly, GQ unfolding times by RPA were also similar for all constructs, 0.2 - 0.3 sec, despite significant differences in their equilibrium stability.