A hairpin conformation for the 3′ overhang of Oxytricha nova telomeric DNA

被引:28
作者
Laporte, L [1 ]
Thomas, GJ [1 ]
机构
[1] Univ Missouri, Sch Biol Sci, Div Cell Biol & Biophys, Kansas City, MO 64110 USA
关键词
telomere; DNA structure; Hoogsteen G center dot G pairs; Raman; spectra; hydrogen exchange;
D O I
10.1006/jmbi.1998.1938
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The solution secondary structure of the Oxytricha nova telomeric 3' over-hang, d(T(4)G(4))(2), has been investigated by Raman spectroscopy, hydrogen-deuterium exchange kinetics and gel electrophoresis. The electrophoretic mobility of d(T(4)G(4))(2) in non-denaturing gels indicates a highly compact conformation, consistent with a hairpin secondary structure. Raman markers show that the d(T(4)G(4))(2) hairpin contains equal numbers of C2'-endo/syn and C2'-endo/anti deoxyguanosine conformers, as well as G.G basepairs of the Hoogsteen type. The hydrogen-deuterium exchange kinetics of d(T(4)G(4))(2), monitored by time-resolved Raman spectroscopy, reveal two kinetically distinct classes of guanine imino (N1H) protons. The more slowly exchanging fraction (k(N1H)((1)) = 4.6 x 10(-3) min(-1)), which represents 50% of N1H groups, is attributed to Hoogsteen-paired residues. The more rapidly exchanging fraction (k(N1H)((2)) greater than or equal to 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(T(4)G(4))(2) reveals modest retardation vis-a-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, dT(6)(T(4)G(4))(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. (C) 1998 Academic Press.
引用
收藏
页码:261 / 270
页数:10
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