Structural Basis of DNA Recognition and Mechanism of Quadruplex Formation by the β Subunit of the Oxytricha Telomere Binding Protein,

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Abstract
Interactions of the β subunit of the Oxytrichanova telomere binding protein with the telomeric DNA sequences, d(T4G4)2 and dT6(T4G4)2, have been investigated in vitro using Raman and fluorescence spectroscopies. Raman difference spectra show that the β subunit binds to both d(T4G4)2 and dT6(T4G4)2 but promotes the formation of a parallel-stranded quadruplex only in dT6(T4G4)2, thus demonstrating the importance of the telomeric 5‘ tail for in vitro recognition and guanine quadruplex formation. While d(T4G4)2 is not a suitable substrate for quadruplex promotion by the β subunit, the Raman spectra reveal other structural rearrangements of this DNA strand upon β subunit binding, including changes in guanine glycosyl torsion angles from syn to anti and disruption of carbonyl hydrogen-bonding interactions. The conformation of d(T4G4)2 in the β:d(T4G4)2 complex is suggested as a plausible intermediate along the pathway to formation of the parallel-stranded guanine quadruplex. Fluorescence band shifts indicate that at least one of the two tryptophans of the β subunit is shielded from solvent as a consequence of DNA binding in both the β:dT6(T4G4)2 and β:d(T4G4)2 complexes. However, the Raman spectra of these complexes suggest no significant changes in the β subunit secondary structure attendant with DNA binding. A model for β subunit binding by Oxytricha telomeric DNA sequences and a mechanism for quadruplex formation are proposed. A key feature of this model is the use of a telomeric hairpin secondary structure as the recognition motif.