Allosteric interaction of minor groove binding ligands with UL9-DNA complexes

The herpes simplex virus type I origin binding protein (UL9) is a sequence- specific DNA binding protein. Several studies have demonstrated that UL9 bi nds to the 11-base pair sequence 5'-CGTTCGCACTT-3' primarily, or solely, th rough interaction with the major groove. Minor groove binding ligands, such as distamycin, netropsin, and GLX, an indole-linked dimer of netropsin, ca n effectively disrupt the UL9-DNA complex only when their DNA binding sites are coincident with the right side of the DNA binding site of the protein and overlap with the protein binding site by two (TT) base pairs. These res ults suggest that the right side of the UL9-DNA complex has a unique struct ure that is sensitive to minor groove ligand binding. In addition, a biphas ic displacement curve was observed with GLX, which suggests two modes of li gand binding which have different effects on UL9-DNA complexes. Using a flu orescence-based hybridization stabilization assay, we determined that GLX c an bind to its binding site as an overlapping dimer (i.e., 2:1 stoichiometr y). Footprinting of UL9-DNA complexes with the minor groove directed chemic al nuclease 1,10-phenanthroline copper confirms that the DNA conformation a t the position of the right-side ligand binding site of GLX is altered and has a widened minor groove. In contrast, it is well established that at 1: 1 stoichiometries, AT sequence specific ligands, such as netropsin, distamy cin, and GLX, prefer uniform, narrow minor grooves. The opposing conformati onal requirements of UL9 and lower concentrations of GLX at the ligand bind ing A-tract overlapping the right side of the protein binding site indicate that allosteric inhibition, rather than direct steric competition, contrib utes to ligand-induced protein displacement. At higher GLX concentrations, giving 2:1 binding in a widened minor groove, co-binding with UL9 is allowe d. A model is presented that is consistent with these observations, and imp lications for targeted regulation of gene transcription are discussed.