Bacteriophage T4 endonuclease II: concerted single-strand nicks yield double-strand cleavage

In vivo, endonuclease II (EndoII) of coliphage T4 cleaves sites with conserved sequence elements (CSEs) to both the left and the right of the cleaved bonds, 16 bp altogether with some variability tolerated. In vitro, however, single-strand nicks in the lower strand predominate at sites containing only the left-side CSE that determines the precise position of lower strand nicks. Upper strand nick positions vary both in vivo and in vitro. A 24 bp substrate was nicked with the same precision as in longer substrates, showing that the conserved sequence suffices for precise nicking by EndoII. Using DNA ligase in vitro, we found that EndoII nicked both strands simultaneously at an in vivo-favoured site but not at an in vitro-favoured site. This indicates that the right-side CSE at in vivo-favoured sites is important for simultaneous nicking of both strands, generating double-strand cleavage. Separate analysis of the two strands following in vitro digestion at two in vitro-favoured sites showed that EndoII nicked the lower strand about 1.5-fold faster than the upper strand. In addition, the upper and lower strands were nicked independently of each other, seldom resulting in double-strand cleavage. Thus, cleavage by EndoII is the fortuitous outcome of two separate nicking events.