Dynamic light-scattering studies of internal motions in DNA. III. Evidence for titratable joints associated with bound polycations.

Abstract

Dynamic light-scattering techniques are employed to study the internal Brownian motions of a commercial calf thymus DNA, clean and contaminated ϕ29 DNAs, and a clean ϕ29 DNA with bound spermidine as a function of pH. The Rouse-Zimm model parameters of both calf thymus and contaminated ϕ29 DNAs differ substantially from those of clean ϕ29 DNA in the neutral-pH region. However, this difference is largely removed by adding 0.01M EDTA (which has no effect on clean ϕ29 DNA) to the calf thymus DNA sample. These findings imply the existence in that preparation of polycation contaminants, presumably basic proteins, that can substantially alter the local mechanical properties of the DNA near their binding sites. The internal motion parameters kB T/f and b of both calf thymus and contaminated ϕ29 DNAs are found to exhibit pronounced characteristic variations between pH 8.5 and 10.5, over which range there is essentially no detectable titration to a resolution of about 1% of the base pairs. These variations, which are not observed for clean ϕ29 DNA, are qualitatively similar to those previously reported for a ϕ29 DNA with 21 single-strand breaks per chain. This indicates the formation of titratable joints associated with bound polycation contaminants. These basic ligands presumably facilitate local denaturation by stabilizing the titration of one or more protons on base-ring nitrogens near their binding sites. Spermidine binding up to 85-87% of neutralization of the total DNA charge has only a relatively minor effect on the internal motion parameters at neutral pH in 0.01M NaCl. However on raising the pH to 10.2, the internal motion parameter kB T/f undergoes a marked decrease similar to that observed for both calf thymus and contaminated ϕ29 DNAs and also ϕ29 DNA with single-strand breaks. This indicates that spermidine, too, is capable of inducing titratable joints. Evidence is presented that the titratable joints associated with bound polycations on the calf thymus DNA may serve primarily as torsion joints, as was found previously for the titratable joints associated with single-strand breaks.