Intramolecular G-quadruplex Unwinding And Kinetic Control Of Human Telomere G-quadruplex Folding

Many of putative quadruplex sequences have been found to exist in essential regions of chromosomes, for example, telomeres, promoter of oncogenes, and the insulin regulatory regions. G-quadruplex structures are believed to play roles in important biological processes, such as regulation of gene expression and maintenance of genome stability. How G-quadruplexes affect the processing of DNA and RNA in various physiological events, such as transcription and translation, is an important question deserving exploration. We established a fluorescent method to monitor the kinetics of intramolecular G-quadruplex unwinding in real-time. Using this method they studied the unwinding of intramolecular G-quadruplex from the human telomere, ILPR and PSMA4sequences by the BLM642-1296helicase and compared it with the unwinding of their corresponding duplex substrates. Our study revealed that the unwinding of the G-quadruplex structures is significantly less efficient than that of the duplexes. In addition, our data show that the efficiency of G-quadruplex unwinding reversibly correlates with quadruplex stability. These facts imply that G-quadruplex can create a barrier to processes such as protein translocation on nucleic acids and can modulate the opening/annealing of genomic DNA.G-quadruplexes are highly polymorphic in their folding topology. The control of G-quadruplex folding and its physiological relevance is not only important for understanding the in vivo function of a G-quadruplex, but also for developing effective drugs. The vast majority of studies have focused on DNA structures in the thermodynamic equilibrium state. However, many biological processes that are essential in gene expression, such as chromatin remodeling and transcriptional control, are mostly regulated by kinetic control. We have studied the kinetic versus thermodynamic control of G-quadruplex folding by using human telomere DNA as a model system. By employing circular dichroism, fluorescence spectroscopy, gel electrophoresis, and photo-cleavage footprinting, we show that when liberated from the DNA duplex upon duplex disruption or helicase unwinding, human telomere DNA initially forms a distinct structure from the one it forms at the equilibrium state in a K+/PEG solution. This result demonstrates that the conformation initially adopted by a G-quadruplex in physiological events may not be the one that it adopts in the equilibrium state, and that the competition between kinetic and thermodynamic control is an important factor in determining the biological and pharma-ceutical relevance of a G-quadruplex.