Alteration of A.T base-pair opening kinetics by the ammonium cation in DNA A-tracts.
We have investigated by NMR the effects of NH(4)(+) 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(GCA(4)T(4)GC)](2), and of a B-DNA duplex,[d(CGCGATCGCG)](2). Upon NH(4)(+) addition to [d(CGCGAATTCGCG)](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(GCA(4)T(4)GC)](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 NH(4)(+) binding in the minor groove. The cross-peak intensities of the NOESY spectra collected at low and high NH(4)(+) 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 NH(4)(+) binding on the A.T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH(4)(+) concentrations show the occurrence of two native conformations in proportions depending on the NH(4)(+) 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 NH(4)(+) 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 NH(3) 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).
D000241:Adenosine / Q000737:chemistry*; D020029:Base Pairing; D002384:Catalysis; D002414:Cations, Monovalent / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D003854:Deoxyribonucleotides / Q000737:chemistry; D007097:Imines / Q000737:chemistry; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D011061:Poly A / Q000737:chemistry*; D011522:Protons; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D013936:Thymidine / Q000737:chemistry*
D002414:Cations, Monovalent; D003854:Deoxyribonucleotides; D007097:Imines; D009692:Nucleic Acid Heteroduplexes; D011522:Protons; D000644:Quaternary Ammonium Compounds; D011061:Poly A; D004247:DNA; D000241:Adenosine; D013936:Thymidine