Preferential location of bulged guanosine internal to a G.C tract by 1H NMR.
A series of double-helical oligodeoxyribonucleotides of sequence corresponding to a frame-shift mutational hot spot in the lambda CI gene, 5'-dGATGGGGCAG, are compared by proton magnetic resonance spectroscopy at 500 MHz of the exchangeable protons. Duplexes containing an extra guanine in a run of two, three, and four G.C base pairs are compared to regular helices of the same sequence and to another sequence containing an isolated bulged G, 5'-dGATGGGCAG.dCTGCGCCATC. The imino proton resonances are assigned by one-dimensional nuclear Overhauser effect spectroscopy. Resonances assigned to the G tract in bulge-containing duplexes are shifted anomalously upfield and are very broad. Imino proton lifetimes are determined by T1 inversion-recovery experiments. The exchange rates of G-tract imino protons in bulged duplexes are rapid compared to those in regular helices and are discussed in terms of the apparent rate of solvent exchange for the isolated G bulge. Delocalization of a bulged guanosine in homopolymeric sequences can explain the observed changes in chemical shift and relaxation times across the entire G.C run, and the chemical shifts can be fit by a simple model of fast exchange between base-paired and unpaired states for the imino protons. This allows us to calculate the relative occupancies of each bulge site. In these sequences, we find the extra base prefers positions internal to the G tract over those at the edge.
Woodson
S A
SA
Department of Chemistry, Yale University, New Haven, Connecticut 06511.
Crothers
D M
DM
D010582:Bacteriophage lambda / Q000235:genetics; D001482:Base Composition; D003596:Cytosine; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D006147:Guanine; D006151:Guanosine; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009154:Mutation; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides
D009838:Oligodeoxyribonucleotides; D006151:Guanosine; D006147:Guanine; D003596:Cytosine