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BsrDI and BtsI restriction endonucleases recognize and cleave double-strand DNA at the sequences GCAATG (2/0) and GCAGTG (2/0),respectively.We have purified and partially characterized these two enzymes,and analyzed the genes that encode them.BsrDI and BtsI are unusual in two respects:each cleaves DNA as a heterodimer of one large subunit and one small subunit; and,in the absence of their small subunits,the large subunits behave as sequence-specific DNA nicking enzymes and only nick the bottom strand of the sequences at these respective positions:GCAATG (-/0) and GCAGTG (-/0).Amino acid sequence comparisons reveal that BsrDI and BtsI belong to a family of restriction enzymes that possess two catalytic sites:a canonical PD-Xn-EXK and a second non-canonical PD-Xn-E-X12-QR.