Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which facilitate the rearrangement of nucleic acid secondary structure. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) have been extensively studied, and nucleic acid aggregation, duplex destabilization, and rapid protein binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as ORF1p from the retrotransposon LINE-1, are not as well understood. We used single molecule DNA stretching in combination with site-directed mutagenesis of ORF1p as a method for detailed characterization of its chaperone activity. Wild type ORF1p significantly reduces the cooperativity of the force-induced melting transition from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA), indicating that DNA melting is more easily initiated in its presence. ORF1p also aggregates both dsDNA and ssDNA, and exhibits relatively rapid binding kinetics. Altering certain residues has dramatic effects on chaperone activity. Stretching curves in the presence of mutant R284A, which is inactive in retrotransposition assays, exhibit a cooperative melting transition and minimal DNA aggregation. Retrotransposition is partially restored with mutant R284K, which alters the melting transition and strongly aggregates ssDNA. Similarly, mutant Y318A has minimal retrotransposition activity, and stretching curves reflect only a small change in melting transition cooperativity. The Y318F mutant, which largely restores retrotransposition, alters the melting transition in a way similar to wild type ORF1p. Thus, DNA stretching results indicate that reduced cooperativity of the melting transition is associated with greater nucleic acid chaperone and retrotransposition activity of ORF1p variants.