Effect of Adenine Methylation on the Structure and Dynamics of TpA Steps in DNA: NMR Structure Determination of [d(CGAGGTTTAAACCTCG)]2 and Its A9-Methylated Derivative at 750 MHz
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- 作者：Jody Lingbeck ; Mark G. Kubinec ; Julie Miller ; Brian R. Reid ; Gary P. Drobny ; and Michael A. Kennedy
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：January 23, 1996
- 年：1996
- 卷：35
- 期：3
- 页码：719 - 734
- 全文大小：2149K
- 年卷期：v.35,no.3(January 23, 1996)
- ISSN：1520-4995
文摘
At TpA steps in DNA, the adenine base experiencesexceptionally large amplitude (20
-50)and slow (10 ms-1 
s) motion [Kennedy et al. (1993)Biochemistry 32, 8022-8035] which hasbeencorrelated with transitions between multiple conformational states[Lefevre et al. (1985) FEBS Lett.190,37-40]. The base dynamics can be detected in one-dimensional1H NMR spectra as excess line widthof the aromatic proton resonances. The magnitude of the excessline width is temperature dependent andreaches a maximum at some temperature. In order to betterunderstand the origin of the dynamics, wehave studied the effect of N6-methylation of the TpAadenine on both the line widths and its local structure.Here, solution-state 500 and 750 MHz 1H NMR datacollected on [d(CGAGGTTTAAACCTCG)]2 showthat the excess line width of theTpA adenine-H2 is diminished when theTpA adenine is N6-methylatedand that the line width no longer experiences a maximum as thetemperature is varied. The resonancessharpen upon methylation because the amplitude of base motion isrestricted due to steric effects and dueto other structural changes at the TpA site. Additionally, boththe TpA adenine-H8 and the exchangeableimino resonance of thymine at the TpA step were also found to haveexcess line width that is diminishedupon N6-methylation. In order to elucidate thestructural features responsible for TpA base dynamics,solution-state NMR structures of [d(CGAGGTTTAAACCTCG)]2and its A9 N6-methylated derivativewere determined at 750 MHz. Comparison of the structures showsthat poor base stacking at the TpAstep may contribute to, or be the origin of, its basedynamics.
-50)and slow (10 ms-1 s) motion [Kennedy et al. (1993)Biochemistry 32, 8022-8035] which hasbeencorrelated with transitions between multiple conformational states[Lefevre et al. (1985) FEBS Lett.190,37-40]. The base dynamics can be detected in one-dimensional1H NMR spectra as excess line widthof the aromatic proton resonances. The magnitude of the excessline width is temperature dependent andreaches a maximum at some temperature. In order to betterunderstand the origin of the dynamics, wehave studied the effect of N6-methylation of the TpAadenine on both the line widths and its local structure.Here, solution-state 500 and 750 MHz 1H NMR datacollected on [d(CGAGGTTTAAACCTCG)]2 showthat the excess line width of theTpA adenine-H2 is diminished when theTpA adenine is N6-methylatedand that the line width no longer experiences a maximum as thetemperature is varied. The resonancessharpen upon methylation because the amplitude of base motion isrestricted due to steric effects and dueto other structural changes at the TpA site. Additionally, boththe TpA adenine-H8 and the exchangeableimino resonance of thymine at the TpA step were also found to haveexcess line width that is diminishedupon N6-methylation. In order to elucidate thestructural features responsible for TpA base dynamics,solution-state NMR structures of [d(CGAGGTTTAAACCTCG)]2and its A9 N6-methylated derivativewere determined at 750 MHz. Comparison of the structures showsthat poor base stacking at the TpAstep may contribute to, or be the origin of, its basedynamics.