Molecular mechanism of DNA recognition by the a subunit of the Oxytricha telomere binding protein

Interactions between telomeric DNA and the a subunit of the heterodimeric t elomere binding protein of Oxytricha nova have been probed by Raman spectro scopy, CD spectroscopy, and nondenaturing gel electrophoresis. Telomeric se quences investigated include the Oxytricha 3' overhang, d(T(4)G(4))(2), and the related sequence dT(6)(T(4)G(4))(2), which incorporates a 5'-thymidyla te leader. Corresponding nontelomeric isomers, d(TG)(8) and dT(6)(TG)(8), h ave also been investigated. Both d(T(4)G(4))(2) and dT(6)(T(4)G(4))(2) form stable hairpins that contain Hoogsteen G.G base pairs [Laporte, L., and Th omas, G. J., Jr. (1998) J. Mel. Biol. 281, 261-270]. The or subunit binds s pecifically and stoichiometrically to the dT(6)(T(4)G(4))(2) hairpin and al ters its secondary structure by inducing conformational changes in the 5'-t hymidylate leader without extensive disruption of G.G base pairing. Convers ely, binding of the alpha subunit to d(T(4)G(4))(2) eliminates G.G pairing and unfolds the hairpin. DNA unfolding is accompanied by conformational cha nges affecting both the backbone and dG residues, as evidenced by Raman and CD spectra. Interestingly, the alpha subunit also forms complexes with the nontelomeric isomers, d(TG)(8) and dT(6)(TG)(8) evidenced by altered elect rophoretic mobility in nondenaturing gels; however, Raman and CD spectra of complexes of the or subunit with nontelomeric DNA suggest no significant c hanges in backbone or deoxynucleoside conformations. Similarly, the alpha s ubunit binds to but does not appreciably alter the secondary structure of d uplex DNA. The present results show that while the alpha subunit has the ca pacity to bind to Watson-Crick and different non-Watson-Crick motifs, DNA r efolding is specific to the Oxytricha telomeric hairpin and the retention o f G.G pairing is specific to the telomeric sequence incorporating the 5' le ading sequence. A model is proposed for alpha subunit binding to telomeric DNA, and the physiological role of the a subunit in telomere organization i s discussed.