Involvement of Coprinus endonuclease in preparing substrate for in vitro recombination

A functional recombination assay involving the tetracycline mutant plasmids, pUW1 and pUW4, was used to assess (i) the nature of the DNA substrates needed and (ii) the involvement of Coprinus endonuclease in preparing substrate, for the RecA-directed recombination process. A gapped circular plasmid and a linear or a nicked circular plasmid are efficient substrate combinations in this system to achieve a 160-fold increase in the in vitro recombination frequency over the control levels. The Coprinus endonuclease obtained from early meiotic prophase can produce such substrates. The recombination frequency obtained with the combination of gapped pUW1 plasmids initially relaxed by the Coprinus endonuclease and linear pUW4 plasmids produced by the site-specific BamHI digest is 10-fold lower than that obtained when both substrates are digested by BamHI. The results suggest that the Coprinus endonuclease creates random nicks on plasmid DNA. Glyoxal gel electrophoretic analysis was used to confirm this random nicking activity of Coprinus endonuclease.