Purα is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Purα unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Purα are essential for both ss- and duplex DNA binding. Purα binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Purα since removal of Purα in the gel eliminates the series and since Purα binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Purα binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Purβ lacking the Purα C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Purα can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Purα.