Abstract: The DNA of the centromere of chromosome IV (CEN4) of Saccharomyces cerevisiae is found to be sensitive to single-strand specific nuclease P1 when inserted into a negatively supercoiled plasmid. Fine mapping identifies two P1-sensitive segments: one segment maps to essential centromere element CDEI and bordering CDEII bases, and the other segment is located in element CDEIII. The AT-rich element CDEII, which is expected to be early melting, is for the most part resistant to nuclease P1. Cleavage is inhibited by NaCl, MgCl2 and polyamines. The cleavage rate is only weakly dependent on P1 concentration in the range of 0.5 to 20 munits/microliters. The two P1-sensitive segments are also modified by the DNA-confirmation-specific reagent KMnO4. Negative superhelicity is required for all modifications. Two-dimensional topoisomer analysis indicates the unwinding of 80(+/- 10) bases within the negatively supercoiled CEN4-containing plasmid. The data best fit a model in which the DNA of the CEN4 region undergoes a transition into a paranemic intermediate in which each strand is folded into an RNA-like foldback structure.