Common fragile sites are chromosomal loci prone to breakage and rearrangement that can be induced by aphidicolin, an inhibitor of DNA polymerases. Within these loci, sites of preferential DNA breaks were proposed to correlate with peaks of enhanced DNA flexibility, the function of which remains elusive. Here we show that mammalian DNA replication origins are enriched in peaks of enhanced flexibility. This finding suggests that the search for these features may help in the mapping of replication origins, and we present evidence supporting this hypothesis. The association of peaks of flexibility with replication origins also suggests that some origins may associate with minor levels of fragility. As shown here, an increased sensitivity to aphidicolin was found near two mammalian DNA replication origins.

We analyzed the replication pattern and the topological organization of a 200 kb long Chinese hamster polygenic locus, which spans the boundary of two isochores. One of them is G C rich while the second one is highly A T rich. Previous analysis of mutants ampli®ed for this locus had identi®ed, within the A T rich isochore, a mitotic recombination hotspot and a replication origin separated by some 7 kb. The recombination hotspot exhibits structural features repeatedly observed at common fragile sites, including a typical enrichment in peaks of enhanced DNA helix exibility. By studying the replication pattern of the same locus in the non-ampli®ed CHO cells, we con®rm here the localization of the replication origin and show that the mitotic recombination hotspot does not correspond to a replicon junction. This ®nding makes questionable current hypotheses correlating replication termination regions with recombination prone sequences. Using topoisomerase II-mediated DNA cleavage at matrix attachment sites, we identi®ed a 40 kb-long DNA anchorage region extending all along a transcription unit nested within the A T rich isochore. Both the recombination hotspot and the replication origin lie within this topoisomerase II sensitive region, which suggests that features essential for initiation of recombination and initiation of DNA replication cluster within DNA anchorage regions. Features common to this region and to common fragile sites are discussed.

The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions.