G4-resolvase 1 tightly binds and catalytically unwinds unimolecular G-quadruplex structures with implications for gene transcription and telomere biology

G-quadruplexes (G4s) are four-stranded structures formed by guanine-rich DNA or RNA sequences in which the strands can be orientated in parallel, antiparallel, or mixed parallel/antiparallel orientations. It has been previously shown that the DHX36 gene product, G4-Resolvase1 (G4R1), binds parallel tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here we show that G4R1 specifically binds to a parallel, unimolecular G4-DNA with remarkable affinity, with Kd values in the low pM range; the tightest reported binding affinity for any known G4-binding protein. Using a novel peptide nucleic acid (PNA) trap assay, we show that G4R1 catalyzes unwinding of unimolecular G4-DNA into an unstructured state. Additionally, we provide strong evidence for the presence of unimolecular G4-structures in the promoter of the Yin Yang 1 (YY1) gene and demonstrate that these structures have an inhibitory effect on reporter assay expression. We also discovered that G4R1 directly binds and unwinds G4-structures in the YY1 promoter, subsequently enhancing reporter expression. Consistently, ectopically expressed G4R1 increased endogenous YY1 levels and gene array analysis consisting of 258 patient breast cancer samples indicated a significant, positive correlation between G4R1 and YY1 expression. We went on to demonstrate that G4R1 tightly binds to mixed parallel/antiparallel and antiparallel unimolecular telomeric G4-DNA. Furthermore, we show specific requirements of G4R1 for tight binding to telomeric DNA which are: 1) presence of G4-structure in the sequence and 2) a guanine-containing 3'-tail. Overall findings from this dissertation strongly suggest that G4R1 tightly and specifically binds to an exceptional array of G4-structures and could potentially serve as a "pan" G4-resolver in the cell. Indeed this finding could have significant genomic impact, as >375,000 G4-forming motifs exist in the genome and G4-structures have been shown to play prominent regulatory roles in transcription, translation, replication, immunoglobin switching, and telomere biology.