R1 restriction endonuclease cleaves duplex DNA at a specific sequence, probably 6 nucleotide pairs in length, by making two single-strand staggered cleavages, generating 5′-phosphoryl and 3′-hydroxyl termini. The single-strand ends produced at each break have identical and complementary sequences of 4 or 6 nucleotides in length. Therefore, the cleavage site possesses a 2-fold rotational axis of symmetry perpendicular to the helix axis. The ends of full-length linear SV40 DNA, generated by R1 endonuclease cleavage, can be joined by Escherichia coli ligase to regenerate duplex, fully infectious, covalently-closed circular molecules. It was further found that all R1 endonuclease-generated ends are identical and complementary. Therefore, any two DNA molecules with R1 sites can be "recombined" at their restriction sites by the sequential action of R1 endonuclease and DNA ligase to generate hybrid DNA molecules.