Telomeres, the ends of linear chromosomes, typically consists of tandem repeats of a simple guanine-rich sequence. Telomeric DNA is able to form intermolecular G-quartet structures. The beta-subunit of the Oxytricha telomere-binding protein acts as a molecular chaperone to promote the formation of dimers and specific higher order complexes of telomeric DNA stabilized by G-quartets; these reactions occur under physiological conditions in vitro. In the present article, we show that, at saturating protein concentrations (> or = 200 nM), beta-mediated G-quartet formation is a first-order reaction with respect to DNA concentration, with k approximately 1 h-1 at 37 degrees C. In contrast, the protein-independent reaction is a second-order reaction. The beta-subunit enhances the rate of G-quartet formation by 10(5)-10(6)-fold at a telomeric DNA concentration of 20 nM. The beta-mediated higher order complexes are identified as parallel four-stranded tetramers of telomeric DNA (G4-DNA). Poly-L-lysine also promotes formation of the tetramers, but not dimers. These DNA structures were studied by irreversible thermal melting experiments and probed by annealing to different complementary strands. Guanine residues important for structure formation were analyzed by methylation interference experiments. On the basis of these data, models for the beta-mediated structures are proposed, and possible mechanisms for the beta-mediated reaction are discussed. In addition, we found that the beta-subunit promotes the annealing of two complementary strands into a duplex, as do many other basic proteins. However, not all proteins with annealing-promoting activity are active in the formation of G-quartet structures. The activity of the telomere protein in promoting the formation of telomeric DNA structures may enable chromosome-chromosome association or the regulation of telomerase activity in vivo.