Hexaamminecobalt(III) binding environments on double‐helical DNA

Previous cation nmr evidence suggests that univalent cations such as Na + bind to DNA in a diffuse, nonspecific manner, whereas di‐ and trivalent cations show distinct binding heterogeneity. Here are reported 59 Co‐ and 23 Na‐nmr measurements of the %GC dependence of the DNA binding behavior of the trivalent hexaamminecobalt(III) cation. When Co(NH 3 ) 6 Cl 3 titrations are performed on one mammalian and three bacterial DNAs, evidence is found for at least three distinct classes of bound Co(NH 3 ) 6 3+ . A comparison of titration curves for all four DNAs demonstrates that an increase in GC content correlates with an increase in the fraction of specific Co(NH 3 ) 6 3+ . binding sites. For M. lysodeikticus DNA (72% GC), a slowly exchanging class of bound 59 Co(NH 3 ) 6 3+ is apparent. This class of sites is saturated at very low binding densities (between 0.02 and 0.03 cobalt cations per DNA phosphate). At higher binding densities (greater than 0.03), the signal due to slowly exchanging 59 Co(NH 3 ) 6 3+ disappears into the noise, and a single 59 Co(NH 3 ) 6 3+ signal is observed. Within the sensitivity limitations of these measurements, no evidence for slowly exchanging bound 59 Co(NH 3 ) 6 3+ could be found for any of the other DNAs, for which a single, rapidly exchanging 59 Co(NH 3 ) 6 3+ signal is observed at all binding densities. For this rapidly exchanging signal, for all four DNAs, the measured 59 Co(NH 3 ) 6 3+ nmr parameters depend significantly on (a) binding density and (b) GC content of the DNA. Since under the low salt conditions of these experiments greater than 90% of the Co(NH 3 ) 6 3+ in solution is localized near the DNA surface [W. H. Braunlin, C. F. Anderson, and M. T. Record, Jr. (1987) Biochemistry , Vol. 26, p. 7724; G. E. Plum and V. A. Bloomfield (1988) Biopolymers , Vol. 27, p. 1045], these data imply a significant dependence of the bound nmr parameters on both binding density and GC content. Based on these data, it is evident that an increase in GC content correlates with an overall decrease in the mobility of bound 59 Co(NH 3 ) 6 3+ . These 59 Co‐nmr data are fitted to a binding model of two classes of rapidly exchanging bound 59 Co(NH 3 ) 6 3+ [the third class of slowly exchanging bound 59 Co(NH 3 ) 6 3+ is not explicitly considered in this model]. The fractions of “strong” (but rapidly exchanging) cation binding sites that are obtained from the fittings suggest that pairs of neighboring guanine residues may provide favorable locations for cation binding. This notion finds support in simple structural considerations and in examples from the x‐ray crystallographic literature. The picture that obtains is of at least three classes of bound 59 Co(NH 3 ) 6 3+ . The rapidly exchanging bound 59 Co(NH 3 ) 6 3+ consists of a small class of transiently localized Co(NH 3 ) 6 3+ superimposed on a larger class of nonspecifically bound, highly mobile Co(NH 3 ) 6 3+ . The third class of very tightly localized bound 59 Co(NH 3 ) 6 3+ is evident only for the M. lysodeikticus DNA. In contrast, when the 23 Na + ‐nmr behavior is monitored for the same titrations, no significant GC dependence is evident for the motional behavior of the bound sodium cation. These 23 Na results thus provide additional support for the idea of a diffuse, nonspecific DNA binding mode for univalent cations such as Na + . © 1992 John Wiley & Sons, Inc.