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G4-RESOLVASE 1 TIGHTLY BINDS AND CATALYTICALLY UNWINDS UNIMOLECULAR G-QUADRUPLEX STRUCTURES WITH IMPLICATIONS FOR GENE TRANSCRIPTION AND TELOMERE BIOLOGY

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title
G4-RESOLVASE 1 TIGHTLY BINDS AND CATALYTICALLY UNWINDS UNIMOLECULAR G-QUADRUPLEX STRUCTURES WITH IMPLICATIONS FOR GENE TRANSCRIPTION AND TELOMERE BIOLOGY
author
Smaldino, Phillip James
abstract
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.
subject
dhx36
G4 Resolvase 1
G-quadruplex
quadruplex
RHAU
Telomere
contributor
Akman, Steven A (committee chair)
Akman, Steven A (committee member)
Vaughn, James P (committee member)
Hollis, Thomas (committee member)
Scarpinato, Karin D (committee member)
Sui, Guangchao (committee member)
Wang, Yuh-Hwa (committee member)
date
2014-01-15T09:35:36Z (accessioned)
2014-07-15T08:30:09Z (available)
2013 (issued)
degree
Cancer Biology (discipline)
embargo
2014-7-15 (terms)
identifier
http://hdl.handle.net/10339/39141 (uri)
language
en (iso)
publisher
Wake Forest University
type
Dissertation

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