ABSTRACT Regulation of gene transcription by structural interconversions of genomic DNA is an emerging biochemical and genetic paradigm that adds to the already diverse repertoire of eukaryotic gene regulatory mechanisms. The appearance of paranemic structures coincident with changes in gene activity, as well as participation of transcription factors that recognize and bind single-stranded DNA at numerous gene promoters in vivo illustrates the authenticity of this concept and its importance in cellular homeostasis. Despite its acceptance, this concept has been minimally described at the biochemical and biophysical levels, as the means by which sequence-specific single-stranded DNAbinding proteins exert transcriptional influence in double-stranded genomes remains largely undefined. Pur is a sequence-specific single-stranded DNA/RNA-binding protein that acts as a repressor of smooth muscle -actin (SM A) gene transcription, and mRNA translation. SM A is an important cytoskeletal protein that contributes contractile, antimigratory, and nonproliferative functions in smooth muscle. In concert with Pur protein family member Pur , and Y-box protein MSY1, Pur enacts repression of SM A gene expression by interacting with a cryptic cis-regulatory element in the 5’ region of the SM A promoter that has been shown to transiently adopt single-stranded conformations in vivo, and to confer transcriptional activation when trans-activator occupied while in a doublestranded conformation. Downregulation of SM A gene expression has been identified to be a contributing factor to cardiovascular disease progression; therefore a thorough understanding of SM A repression mechanisms is critical for clinical management of these conditions. Although highly homologous at the primary sequence level, Pur and Pur display significant conserved regions of sequence divergence that suggest these paralogs exert distinct cellular functions in various vertebrate classes. A goal of the studies presented herein was to delineate exhibited functional differences with respect to SM A repression in pertinent mouse cell lines. Loss-of-function and chromatin immunoprecipitation studies verified that Pur differs from Pur in that Pur is the dominant Pur protein repressor of SM A expression in embryonic fibroblasts and vascular smooth muscle cells, although by different, cell type-specific mechanisms. Biophysical assessment of Pur single-stranded DNA binding properties showed that despite the ability of Pur to self-dimerize in the absence of nucleic acid, Pur binds to the cryptic SM A enhancer by a sequential and cooperative mechanism, with remarkable affinity and a terminal stoichiometry of 2 to 1. Footprinting and in vitro binding site characterization confirms two Pur binding sites exist within this element and display slight degeneracy from a proposed Pur protein-binding consensus motif. These findings delineate binding mechanisms adopted by Pur and provide a means to identify putative Pur binding sites throughout the genome.