Alteration of A•T base-pair opening kinetics by the ammonium cation in DNA A-tracts

K. Snoussi, Jeal Louis Leroy

Research output: Contribution to journalArticlepeer-review

Abstract

We have investigated by NMR the effects of NH4 + on the chemical shifts, on the structure, and on the imino proton exchange kinetics of two duplexes containing an A-tract, [d(CGCGAATTCGCG)]2 and [d(GCA4T4GC)]2, and of a B-DNA duplex, [d(CGCGATCGCG)]2. Upon NH4 + addition to [d(CGC-GAATTCGCG)]2, the adenosine H2 protons, the thymidine imino protons, and the guanosine imino proton of the adjacent G•C pair show unambiguous chemical shifts. Similar shifts are observed in the A-tract of [d(GCA4T4GC)]2 and for the A5(H2) proton of the B DNA duplex [d(CGCGATCGCG)]2. The localization of the shifted protons suggests an effect related to NH4 + binding in the minor groove. The cross-peak intensities of the NOESY spectra collected at low and high NH4 + concentrations are comparable, and the COSY spectra do not show any change of the sugar pucker. This indicates a modest effect of ammonium binding on the duplex structures. Nevertheless, the imino proton exchange catalysis by ammonia provides evidence for a substantial effect of NH4 + binding on the A•T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH4 + concentrations show the occurrence of two native conformations in proportions depending on the NH4 + concentration. The base-pair lifetimes and the open-state lifetimes of each conformation are distinct. Exchange from each conformation proceeds via a single open state. But if, and only if, the NH4 + concentration is kept larger than 1 M, the A•T imino proton exchange times of A-tract sequences exhibit a linear dependence versus the inverse of the NH3 proton acceptor concentration. This had been interpreted as an indication for two distinct base-pair opening modes (Wärmländer, S., Sen, A., and Leijon, M. (2000) Biochemistry 39, 607-615).

Original languageEnglish (US)
Pages (from-to)12467-12474
Number of pages8
JournalBiochemistry
Volume41
Issue number41
DOIs
StatePublished - Oct 15 2002

ASJC Scopus subject areas

  • Biochemistry

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