The effects of loop size on Sac7d-hairpin DNA interactions.

Hairpin structure is a common feature of DNA molecules. They are located near functional loci, such as regulation and promotion sites, as well as in cruciform structures, and they provide potential binding sites for endogenous proteins. The effects of different hairpin loops that are composed of one to five thymidines, designated as L1-L5, and have a common self-complementary stem, CTATATAG, on the interactions with Sac7d were studied. In thermostability studies, Sac7d stabilized a tetra-loop hairpin DNA and hairpin DNA with GTTC tetra-loop regions better than it stabilized tri- and penta-loops. Circular dichroism (CD) spectra showed that hairpins retained primarily a B-type conformation upon Sac7d binding. Intermolecular interactions between hairpins were likely decreased, due to the Sac7d-induced kinks, as shown by an increase at 220nm in the CD spectra. Surface plasmon resonance (SPR) observations suggested that the rates of Sac7d binding to hairpin DNA depend on the loop size of the hairpin duplexes. At a fixed stem length, Sac7d binds to tetra-loop hairpin DNA duplexes with a higher association rate and lower dissociation rate, compared with their tri- and penta-loop counterparts. In addition, the tri-loop and GTC tri-loop hairpin DNA had lower affinity for Sac7d because of the smaller and tighter loop size. Our study indicates that Sac7d binding affinity to hairpin DNA is primarily determined by loop size and stem integrity, and the results presented here provide a model for studies concerning other minor groove DNA-binding proteins that kink hairpin DNA. Crown