The L2Hs sequences are a polymorphic, interspersed, middle repetitive DNA family unique to human genomes. Genomic fingerprinting indicates that these DNAs vary from one individual to another and between tissues of the same individual. Sequence analysis reveals that they are AT-rich (76%) and contain many unusual sequence arrangements (palindromes, inverted and direct repeats). These sequence properties confer on the L2Hs elements the potential to fold into non-B-form structures, a characteristic of recombination hot spots. To test this hypothesis carbodiimide, osmium tetroxide and S$\sb1$ nuclease were used as single-strand specific probes to study a recombinant plasmid, pN6.4.39, containing a single L2Hs segment. Different forms of the plasmid substrate were analyzed, including linear molecules and circular forms of low, intermediate and high superhelical densities. In addition, plasmid DNA in growing E. coli cells were analyzed. Modified plasmid DNA was analyzed by primer extension in a sequencing-type reaction format. These studies demonstrate that the L2Hs sequences: (1) assume non-B-form structures both in vitro and in vivo, (2) map to predicted cruciform structures, (3) behave as C-type extrusion sequences, and (4) that these unusual DNA structures are dependent on plasmid superhelicity.

A uniquely human interspersed repetitive DNA sequence family, the L2Hs, are highly polymorphic in human genomes. Several features of interspersed repeated DNA may contribute to the instability observed. Certain motifs (direct repeats, palindromes, and inverted repeats) comprising L2Hs elements may adopt unusual secondary structures such as cruciforms or hairpins. These motifs have been associated with features of genome instability in recombination, insertions and deletions. The L2Hs elements also are AT-rich (76%) compared to the bulk of human DNA (52%). That their dynamic nature (i.e. polymorphisms) may arise from recombination, insertions and deletions has led to the hypothesis that the L2Hs element is intrinsically dynamic and may influence the stability of the surrounding genome. Thus, the stability of the L2Hs element was tested in a bacterial model system. A cloned 0.6 kb L2Hs element forms non-B-form structures in recombinant plasmids pN6 and pN2, which differ only in insert orientation. Instability of pN6 and pN2 plasmids was observed in serial propagation studies in which E. coli cells containing the plasmids were cultured every 24 hours for 28 days. The vector plasmid pTZ19U, as control, was found to be stable in all passages while the two L2Hs recombinants developed deletions of the L2Hs insert as well as adjacent vector sequences. The isolated deletion mutants have been characterized via restriction cleavage studies and sequencing to map the boundaries of the deletions. Direct repeats and potential stem-loop structures have been discovered at or within close proximity to the deletion boundaries. The data demonstrate that the L2Hs recombinants' unusual sequence features with potential for non-B-form secondary structures, influence genome stability via their involvement in generating errors during DNA replication and DNA repair.