Unfolding of Non-Telomeric G-Quadruplexes of Varying Stabilities by Replication Protein A

Abstract

We studied the stability of various G-quadruplex (GQ) structures formed by sub-sections of a highly heterogeneous and guanine rich 45 nucleotide long DNA sequence, called TH-12 and located in the promoter region of tyrosine hydroxylase (TH) gene, using single-molecule FRET and conventional biochemistry methods. TH-12 has seven regions that contain two or more consecutive guanines capable of forming various GQs of potentially different stabilities. TH-12 is within a conserved region of TH promoter, is in the immediate vicinity of transcription start site, and is invariably included for maintaining the promoter activity. This work serves as the first step in identifying possible roles that these GQs, with potentially different stabilities, play in regulating TH expression. Abnormality in TH gene expression is associated with a variety of psychiatric problems, including schizophrenia, depression and bipolar disorder and is also linked to Parkinson's disease. We measured the UV-melting temperatures of these GQ structures formed by sub-sections of TH12 and established that some of these GQs are stable against Watson-Crick base pairing, in the presence of a complementary strand, at physiological potassium concentrations. Finally, we studied the viability of these structures against the DNA binding activity of human Replication Protein A (RPA), the most abundant single strand DNA binding protein in eukaryotes. Most GQs were unfolded at RPA concentrations that correlated with their melting temperatures, while a GQ formed by a certain sub-section of TH-12 remained folded even at micromolar RPA concentrations. We propose that this subsection of the DNA sequence is the most likely candidate for forming a physiologically relevant GQ that can be involved in TH regulation. These studies also demonstrate that it is possible to have stable enough GQs that can not be unfolded by RPA activity at physiological ionic strength.