Structural analysis of hemicatenated DNA loops

BACKGROUND: We have previously isolated a stable alternative DNA structure, which was formed in vitro by reassociation of the strands of DNA fragments containing a 62 bp tract of the CA-microsatellite poly(CA)·poly(TG). In the model which was proposed for this structure the double helix is folded into a loop, the base of the loop consists of a DNA junction in which one of the strands of one duplex passes between the two strands of the other duplex, forming a DNA hemicatenane in a hemiknot structure. The hemiknot DNA structures obtained with long CA/TG inserts have been imaged by AFM allowing us to directly visualize the loops. RESULTS: Here we have analyzed this structure with several different techniques: high-resolution gel electrophoresis, probing by digestion with single stranded DNA-specific nucleases or with DNase I, modification with chemicals specific for unpaired bases, and atomic force microscopy. The data show a change in DNA structure localized to the CA/TG sequence and allow us to better understand the structure of this alternative conformation and the mechanism of its formation. CONCLUSIONS: The present work is in good agreement with the model of hemicatenated DNA loop proposed previously. In the presence of protein HMGB1, shifted reassociation of the strands of DNA fragments containing a tract of the poly(CA)·poly(TG) microsatellite leads to the formation of DNA loops maintained at their base by a hemicatenated junction located within the repetitive sequence. No mobility of the junction along the DNA molecule could be detected under the conditions used. The novel possibility to prepare DNA hemicatenanes should be useful to further study this alternative DNA structure and its involvement in replication or recombination.