DNA cruciforms and the nuclear supporting structure

Cruciforms have been suggested as potential recognition structures at or near origins of DNA replication in eukaryotic cells. Monoclonal antibodies with structural specificity for DNA cruciforms have been produced (Frappier et al. J. Mol. Biol. 193, 751, 1987). The effect of these antibodies, when introduced into permeabilized cells, was to increase overall DNA synthesis and relative copy number of genes (Zannis-Hadjopoulos et al. EMBO J. 7, 1837, 1988); this was interpreted to be a consequence of antibody stabilization of the cruciforms located at or near replication origins resulting in multiple initiations of DNA replication at a single site. Fluorescent labeling of nuclei with anti-cruciform antibodies produces a nonuniform pattern of fluorescence in cells arrested at the G1/S boundary which then changes with progression through S-phase (Ward et al. Exp. Cell Res. 188, 235, 1990). In order to determine the relationship of cruciform distribution in DNA with the nuclear matrix/chromosomal scaffold, we assessed the susceptibility of DNA containing cruciforms to digestion with DNase I. The majority of the cruciforms detectable at G1/S and throughout the nucleus are readily digested by DNase, suggesting that cruciform structures may not be intimately associated with matrix proteins. The fraction that is resistant to DNase I appears associated with nuclear membrane and the nucleolus. No cruciforms could be detected in metaphase chromosomes; cruciforms either are not present or are inaccessible--buried in the scaffold. The absence of cruciforms from metaphase chromosomes would be consistent with the viewpoint that the cruciform in vivo is a transient structure dependent upon and interacting with proteins essential for replication or transcription.