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Autonomously replicating segment identified in the murine p53 gene contains p53 recognition sequence and bending region.

The mouse p53 gene, spreading over 16 kb, was divided into 10 segments of approximately 1,000 bp and subcloned into pUC19. The clones were transfected into mouse L cells together with an expression vector of the hygromycin B- or blasticidin S-resistance gene. The transfected cells were cultured in the drug-containing medium to establish cell lines resistant to the drug. Among the established lines, a high percentage of the cells originally transfected with pp53(H-R), possessing the HindIII-EcoRI fragment located downstream from the last coding exon of the p53 gene, harbored plasmid DNAs in an episomal state. The plasmid DNAs recovered from the 53HR cell-lines, transfected with pp53(H-R), were indistinguishable in structure from the original plasmids used for transfection, as assayed by Southern blotting and back-transformation to bacteria. The plasmids in episome of the cells replicated once per cell cycle in S phase in concert with chromosomal DNA. In the HindIII-EcoRI fragment (i.e., the p53(H-R) fragment) which showed a highly efficient replication activity, there exists a putative sequence for p53 recognition near the HindIII site. A DNA bending region with the clusters of AT tracts also exists near the EcoRI site of the same p53(H-R) fragment. In a transient replication system, pp53(H-R) autonomously replicated in episome of transfected cells, while mutant plasmids lacking either the p53 recognition sequence or the bending region did not. The results suggest that the HindIII-EcoRI region downstream from the p53 gene contains an activity of cellular DNA replication and both the p53 recognition sequence and the bending region are necessary for the replication activity.