4616 Nucleic Acids Research, 2007, Vol. 35, No. 14 Figure 7. (A) DNA nicking or restriction activity of BtsI A/B mutants. B cat = catalytic-deﬁcient B subunit. fX174 RF DNA (1 BtsI site) was used as the substrate for nicking or restriction assay. NC, nicked-circular DNA; L, linear DNA; SC, supercoiled DNA. (B) Run-oﬀ sequencing of gel-puriﬁed, nicked-circular DNA. The expected nicking speciﬁcity of Nt.BtsI is GCAGTGNN# (BtsI site and the adjacent sequence are shown at the bottom of the ﬁgure).