Stability and Kinetics of c-MYC Promoter G-Quadruplexes Studied by Single-Molecule Manipulation

Abstract

A DNA G-quadruplex (G4) formed at the oncogene c-MYC promoter region functions as a gene silencer. Due to its high stability at physiological K(+) concentrations, its thermodynamics and kinetic properties have not been characterized in physiological solution conditions. In this work, we investigated the unfolding and folding transitions of single c-MYC G4 and several of its truncated or point mutants at 100 mM KCl concentration under mechanical force. We found that the wild type could fold into multiple species, and the major specie has a slow unfolding rate of (1.4 ± 1.0) × 10(-6) s(-1). The force-dependent thermodynamics and kinetic properties of the major specie were obtained by studying a truncated mutant, Myc2345, that contains the G-tracts 2, 3, 4, and 5. As the c-MYC G4 is a prototype of many other intermolecular parallel-stranded G4's, our results provide important insights into the stability of a broad class of promoter G4's which also play a role in transcription regulation and are potential anticancer targets.

RHAU helicase stabilizes G4 in its nucleotide-free state and destabilizes G4 upon ATP hydrolysis

“…The G4 forming ssDNA sequences were ligated with 5′-thiol labeled 489 bp and 5′-biotin labeled 601 bp dsDNA prepared by polymerase chain reaction as described previously (6,32). The ligated products were purified by gel extraction with PureLink kit (Invitrogen).…”

“…Studies have shown that G4 structures exist in vivo (3,4) where they are involved in multiple biological processes including DNA replication, transcription, translation and telomere maintenance (2). Due to their high thermal and kinetic stability, G4 structures often present a physical barrier to various processes taking place on DNA and RNA (5,6). In vivo , the G4 barrier is overcome by a set of helicases that can unfold G4 structures (7–10).…”

“…Contrary to the unwinding activity of helicases measured in previous bulk assay mainly based on analyzing unwound products (25), our studies are based on the mechanical manipulation of a single G4 molecules using magnetic tweezers. This approach is able to detect the dynamics of G4 unfolding with high spatial and temporal resolutions (6,31). Our results suggest that the ability of RHAU to destabilize G4s is regulated by different nucleotide bound states in an ATPase cycle, and is crucially dependent on the interaction with ssDNA as well as with the G4 specifically through the RSM domain of RHAU.…”

Simple Horizontal Magnetic Tweezers for Micromanipulation of Single DNA Molecules and DNA—Protein Complexes

“…Recent designs allow simultaneous and independent application of forces and torques (13,14), and one report (15) describes a way to decouple twist fluctuations from applied forces. Other developments include a portable version (16), a design with sub-nanometer extension precision (17), a magnetic system for steering beads (18,19), integration of a very high speed camera (20), magnetic micro-manipulation of confined DNA (21), and use of proteins (instead of DNA) as tethers (22). …”

Self-assembly of c-myc DNA promoted by a single enantiomer ruthenium complex as a potential nuclear targeting gene carrier

“…achieved many remarkable results in the field of DNA nano-assembly, which might be applied to potential gene carriers, drug delivery systems, and gene-silencing therapy and cellular imaging3293031. By contrast, evidence has previously showed that the guanine-rich sequence of the c-myc promoter forms G-quadruplex structures because of three or four guanine bases that are associated in a cyclic array via Hoogsteen hydrogen bonding arrangements3233. Studies in our laboratory showed that ruthenium (II) complexes can also facilitate stability of the c-myc G-quadruplex DNA3435.…”