DNA breakage and closure by rat liver type 1 topoisomerase: separation of the half-reactions by using a single-stranded DNA substrate.
Circular single strands of bacteriophage phi X174 DNA are broken by rat liver DNA nicking-closing enzyme (type 1 topoisomerase) in low salt (50 mM KCl) at 37 degrees C, generating linear strands containing covalently bound enzyme [Been, M. D. & Champoux, J. J. (1980) Nucleic Acids Res. 8, 6129-6142]. The linear strands can be recircularized in the presence of 10 mM MgCl2 at 24 degrees C and 37 degrees C or 250 mM KCl at 24 degrees C. Recircularization is blocked when the hydroxyl group at the 5' terminus is phosphorylated. The linears generated by the nicking-closing enzyme can also be joined to other DNA fragments containing 5' hydroxyls, but not 5' phosphates. The linkage formed in both the intrastrand and interstrand reactions is stable to alkali. Reclosure of broken single strands is presumed to be analogous to the closure step that occurs durng nicking and closing cycles on duplex DNA.
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