Reactions of type II restriction endonucleases with 8-base pair recognition sites

Type II restriction endonucleases usually recognize 4-6-base pair (bp) site s on DNA and cleave each site in a separate reaction. A few type II endonuc leases have 8-bp recognition sites, but these seem unsuited for restriction , since their sites are rare on most DNA. Moreover, only one endonuclease t hat recognizes a target containing 8 bp has been examined to date, and this enzyme, SfiI, needs two copies of this site for its DNA cleavage reaction. In this study, several endonucleases with 8-bp sites were tested on plasmi ds that have either one or two copies of the relevant sequence to determine if they also need two sites. SgfI, SrfI, FseI, PacI, PmeI, Sse8781I, and S daI all acted through equal and independent reactions at each site. Asci cl eaved the DNA with one site at the same rate as that with two sites but act ed processively on the latter. In contrast, SgrAI. showed a marked preferen ce for the plasmid with two sites and cleaved both sites on this DNA in a c oncerted manner, like SfiI. Endonucleases that require two copies of an 8-b p sequence may be widespread in nature, where, despite this seemingly inapp ropriate requirement, they may function in DNA restriction.