A hairpin conformation for the 3' overhang of Oxytricha nova telomeric DNA.
The solution secondary structure of the Oxytricha nova telomeric 3' overhang, d(T4G4)2, has been investigated by Raman spectroscopy, hydrogen-deuterium exchange kinetics and gel electrophoresis. The electrophoretic mobility of d(T4G4)2 in non-denaturing gels indicates a highly compact conformation, consistent with a hairpin secondary structure. Raman markers show that the d(T4G4)2 hairpin contains equal numbers of C2'-endo/syn and C2'-endo/anti deoxyguanosine conformers, as well as G.G base-pairs of the Hoogsteen type. The hydrogen-deuterium exchange kinetics of d(T4G4)2, monitored by time-resolved Raman spectroscopy, reveal two kinetically distinct classes of guanine imino (N1H) protons. The more slowly exchanging fraction (kN1H(1)=4.6x10(-3) min-1), which represents 50% of N1H groups, is attributed to Hoogsteen-paired residues. The more rapidly exchanging fraction (kN1H(2)>/=0.3 min-1) is attributable to solvent-exposed residues. Raman dynamic probe of the kinetics of guanine C8H-->C8(2)H exchange in d(T4G4)2 reveals modest retardation vis-Ã -vis dGMP, which rules out quadruplex formation by the telomeric repeat and confirms an ordered secondary structure consistent with a Hoogsteen-paired hairpin. Similar Raman, hydrogen-isotope exchange and electrophoretic mobility experiments on the related telomeric model, dT6(T4G4)2, also reveal a hairpin stabilized by Hoogsteen G.G pairs. Presence of the 5' thymidine tail preceding the Oxytricha telomeric repeat has no apparent effect on the hairpin secondary structure. We propose a molecular model for the hairpin conformation of the Oxytricha nova telomeric repeat and consider its possible roles in mechanisms of telomeric DNA interaction in vitro and telomere function in vivo.[1]References
- A hairpin conformation for the 3' overhang of Oxytricha nova telomeric DNA. Laporte, L., Thomas, G.J. J. Mol. Biol. (1998) [Pubmed]
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