| Abstract: | Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S1 nuclease cleavage. The S1-cleavage occurred once at these sites. In the absence of added Mg2+, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S1, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S1-cleavable, unbasepaired sites. |
