Preparation of G-quartet structures and detection by native gel electrophoresis.

Mounting evidence supporting the existence of DNA structures containing G-quartets in vivo makes these unique and diverse nucleic acid structures an important research subject, and future investigations aimed at elucidating their biological significance are expected. The purification and characterization of G-quartet structures can be challenging because their inherent structural diversity, complexity, and stability are sensitive to an array of variables. The stability of G-quartet structures depends on many factors including number of DNA strands involved in G-quartet formation, the identity of the stabilizing cation(s), the number and sequence context of the guanosines involved in stacking, the presence of single-stranded overhangs, the intervening loop size, and the identity of nucleosides in the loop. Here we detail current methods used in G-quartet preparation and their purification and characterization by native gel electrophoresis.

The human telomere and its relationship to human disease, therapy, and tissue engineering.

The chromosomes of eukaryotes end in a specialized complex of proteins and repetitive DNA called the telomere. Telomeres form a protective cap that prevents chromosome fusions, protects chromosome ends from degradation, and assists in positioning chromosomes in the nucleus. In the absence of replenishing mechanisms, telomeric DNA is lost during each cell cycle owing to incomplete replication, oxidative damage, and nucleolytic degradation. The ribonucleoprotein complex telomerase offsets this loss of telomeric DNA, but its activity is absent in most differentiated human cells. Thus, the aging process results in ever shortening lengths of telomeric DNA. Related to this is the requirement for a mechanism of telomeric DNA maintenance in tumors, leading to telomerase expression in >85% of all cancers cells. The integral roles of telomere biology in these pathophysiological states have substantially motivated its investigation. Here, the literature on the human telomere will be reviewed with an emphasis on the relationship to human health.

Electron microscopic visualization of telomerase from Euplotes aediculatus bound to a model telomere DNA.

Binding of the telomerase ribonucleoprotein from the ciliate Euplotes aediculatus to telomeric DNA in vitro has been examined by electron microscopy (EM). Visualization of the structures that formed revealed a globular protein complex that localized to the DNA end containing the E. aediculatus telomere consensus 3'-single-strand T(4)G(4)T(4)G(4)T(4)G(2) overhang. Gel filtration confirmed that purified E. aediculatus telomerase is an active dimer in solution, and comparison of the size of the DNA-associated complex with apoferritin suggests that E. aediculatus telomerase binds to a single telomeric 3'-end as a dimer. Up to 43% of the telomerase-DNA complexes appeared by EM to involve tetramers or larger multimers of telomerase in association with two or more DNA ends. These data provide the first direct evidence that telomerase is a functional dimer and suggest that two telomerase ribonucleoprotein particles cooperate to elongate each Euplotes telomere in vivo.

Extension of G-quadruplex DNA by ciliate telomerase.

Telomeric DNA can fold into four-stranded structures known as G-quadruplexes. Here we investigate the ability of G-quadruplex DNA to serve as a substrate for recombinant Tetrahymena and native Euplotes telomerase. Inter- and intramolecular G-quadruplexes were gel-purified and their stability examined using native gel electrophoresis, circular dichroism (CD) and thermal denaturation. While intermolecular G-quadruplexes were highly stable, they were excellent substrates for both ciliate telomerases in primer extension assays. In contrast, intramolecular G-quadruplexes formed in K+ exhibited biphasic unfolding and were not extended by ciliate telomerases. Na+-stabilised intramolecular G-quadruplexes were extended by telomerase owing to their rapid rate of dissociation. The Tetrahymena telomerase protein component bound to inter- but not intramolecular K+-stabilised G-quadruplexes. This study provides evidence that parallel intermolecular G-quadruplexes can serve as substrates for telomerase in vitro, their extension being mediated through direct interactions between this higher-order structure and telomerase.

Nucleic acid-binding ligands identify new mechanisms to inhibit telomerase.

We screened a small library of known nucleic acid-binding ligands in order to identify novel inhibitors of recombinant human telomerase. Inhibitory compounds were classified into two groups: Group I inhibitors had a notably greater effect when added prior to telomerase assemblage and Group II inhibitors displayed comparable inhibition when added before or after telomerase assemblage. Hoechst 33258, a Group I inhibitor, was found to interact tightly (KD = 0.36 microM) with human telomerase RNA (hTR) leading us to propose that hTR is the molecular target for this and other Group I inhibitors. Our results suggest that hTR can be exploited as a small-molecule drug target and provide several new structural motifs for the further development of novel telomerase inhibitors.