Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease.
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.
D001412:Bacillus subtilis; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D015719:Single-Strand Specific DNA and RNA Endonucleases
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004264:DNA Topoisomerases, Type I; D008274:Magnesium