DNA INVERTED REPEATS AND HUMAN DISEASE

John J. Bissler

The Children's Hospital Research Foundation, Cincinnati, Ohio

5. IN VIVO FORMATION OF CRUCIFORMS

The DNA features that cause cruciform extrusion in vitro have been demonstrated to also lead to alternative secondary structures in vivo (13). The basic approaches used to study cruciforms have been to demonstrate the extruded hairpin by chemically modifying the single-stranded loop, having protein recognition of the extrusion itself, or by chemically cross-linking bases in the cruciform stem.

5.1. Chemical Modification of the Hairpin Loop

Chemical reactivity of the bases is influenced by base pairing. Single-strand bases, such as in hairpin loops, have different reactivity than in duplex DNA. Electrophiles like osmium tetroxide have been used to identify the extruded hairpin loop at basepair resolution (14-16). These sites can be detected by strategies employing either primer extension, because DNA polymerization arrests at the modified site, or by cleavage of the phosphodiester backbone at these sites by piperidine. Following modification of most bases, the glycosidic bond is more readily hydrolyzed resulting in an abasic site.

5.2. Protein Recognition of the Extruded Hairpin

5.2.1. Antibody Studies

An immunologic approach also has been employed using antibodies that recognize the sequence independent structure of the extruded cruciforms (17-20). This approach has identified antibody binding in eukaryotic cells. Ward et. al. successfully used this approach to support the hypothesis that many inverted repeats exist as cruciform structures in vivo (18.)

5.2.2. Endonuclease

Another approach to detect cruciform extrusion in living Escherichia used the enzyme T7 endonuclease VII controlled by an inducible promoter. Induction of the enzyme led to fragmentation of the DNA. The fragmentation implied that cruciforms in the bacterial genome were extruded and therefore were substrates for the enzymatic cleavage (21).

5..3. Chemical Cross-linking

The last and most elegant technique illustrating cruciform formation in vivo employed the bi-functional cross-linking agent 4,5,8-trimethylpsoralen (22, 23). Trimethypsoralen is freely permeable into cells and non-toxic unless activated by 360nm light. Therefore, the DNA structures within the cells are caught in a much more natural state. This approach has identified the extrusion of a well characterized inverted repeat in prokaryotic cells (13). The collective conclusion drawn from these studies indicates that cruciforms can and do form in vivo.