4610 Nucleic Acids Research, 2007, Vol. 35, No. 14 A and B subunits or by mixing puriﬁed BsrDI A and B subunits. IPTG-induced cell extracts were used to digest or pUC19 DNA. Nicking activity was assayed on a single-site substrate pGPS2.1 or pUC-Km (a pUC19 derivative with a KmR gene). Purification of BsrDI A and B subunits Cell extract containing BsrDI A subunit was heated at 658C for 30 min and the denatured E. coli proteins were removed by centrifugation. The BsrDI A subunit in the supernatant was further puriﬁed by chromatography through Heparin Sepharose FF, DEAE Sepharose and Q Sepharose columns (GE HealthCare). Cell extracts containing BsrDI B subunit were prepared from 20 g of IPTG-induced cells. BsrDI B subunit (Nb.BsrDI) was puriﬁed by chromatography through a Heparin Hyper D column (BioSepra), a Q Sepharose column and a Heparin column (Tosoh Bioscience). Cloning of the BtsI R-M system ApoI, NlaIII and Sau3AI partially digested genomic fragments from Bacillus thermoglucosidasius were ligated to EcoRI, SphI and BamHI digested, CIP-treated pUC19, respectively. The primary DNA libraries were challenged by BtsI digestion. After retransformation, plasmids were isolated and screened for resistance to BtsI digestion. The inserts in the resistant plasmids (M+ clones) were sequenced with pUC universal primers and custom-made primers. ORFs adjacent to btsIM genes were obtained by inverse PCR and direct sequencing of the PCR products. The btsIM1 and btsIM2 genes were ampliﬁed in PCR and cloned in two steps into pACYC184 to generate pre-modiﬁed host ER2683 (pACYC-btsIM1M2). The btsIRA and btsIRB genes were ampliﬁed in PCR, digested with appropriate REases and ligated to pUC19 separately and transferred into pre-modiﬁed host ER2683 (pACYC-btsIM1M2). Purification of BtsI A and B subunits BtsI A subunit was puriﬁed by heat denaturation of E. coli proteins and chromatography through a hydroxyapatite column. Cell extracts containing BtsI B subunit were heated at 658C for 30 min and then centrifuged at 26 000 g for 20 min at 48C to remove most of E. coli proteins. BtsI B subunit was puriﬁed by Heparin and hydroxyapatite column chromatography. Reconstitution of BtsI endonuclease activity Cell extracts containing BtsI A (small) and B (large) subunits or puriﬁed A and B subunits were mixed and incubated with 1 mg of fX174 DNA (RF form with a single BtsI site) at 558C for 1 h. The cleavage products were analyzed by agarose gel electrophoresis and com- pared with the cleavage pattern of the native BtsI REase. The nicking activity was also assayed on fX174 ds DNA. To conﬁrm the nicking strand speciﬁcity of the BtsI B subunit, the nicked circular product was subjected to run-oﬀ sequencing. Site-directed mutagenesis of catalytic residues The Asp, Glu and Lys catalytic residues in the active site for top-strand nicking (PDX12–17EXK) and the Asp, Glu, Gln and Arg residues in the active site for bottom-strand nicking (PDX13–18EX12QR) were mutated to Ala residue by site-directed mutagenesis with Phusion DNA polymer- ase and inverse PCR. Following DpnI digestion, plasmids coding for inactive proteins (inverse PCR products) were transformed into methylase-protected host for expression. The entire alleles were sequenced to conﬁrm the desired mutation. Cell extracts were prepared from IPTG-induced cultures. Catalytically inactive A subunit was combined with wild-type (wt) B subunit or catalytic-deﬁcient B subunit was mixed with wt A subunit. Reconstituted restriction or nicking activity was examined using the appropriate DNA substrates. RESULTS Cloning and expression of BsrDI R-M system Several BsrDI-resistant clones were isolated following the methylase selection procedure (14). An M+ clone with a 4-kb insert was sequenced and three potential ORFs were found (Figure 1A). The aa sequence of one ORF was found to resemble a putative DNA transposase. The other two ORFs contain conserved motifs of adenine N6 and cytosine N4 methyltransferases and were named M1.BsrDI and M2.BsrDI, respectively. The two M genes were ampliﬁed in PCR and cloned into the plasmid vector pLG339 to produce pre-modiﬁed host ER2566 (pLG-bsrDIM1M2). After several rounds of inverse PCR and sequencing, three additional ORFs (ORF1, ORF2 and ORF3) were found (Figure 1A). We fortuitously discovered that the gpORF1 is a strand-speciﬁc DNA nicking endonuclease. When IPTG- induced cell extracts containing gpORF1 was used to digest DNA at 658C, no apparent ds DNA cleavage activity was detected. However, when the cell extracts of gpORF1 were incubated with supercoiled pUC19, the DNA was converted to nicked-circular form. The nicking is site speciﬁc and not due to contaminating E. coli nucleases since a pBR322 derivative with BsrDI site deletion was not nicked (data not shown). ORF2 was ampliﬁed in PCR and cloned into pAII17. IPTG-induced cell extracts lack site-speciﬁc restriction or nicking activity (data not shown). The negative result was not due to the absence of the expressed protein since a 25-kDa protein band was clearly detected by SDS–PAGE. In addition, no mutations had been introduced into the cloned insert. Characterization of BsrDI large and small subunit activity We conﬁrmed that both the BsrDI large and small subunits are required for restriction activity. The BsrDI small subunit (A subunit) was partially puriﬁed and its purity was estimated to be 90% by SDS–PAGE analysis (Figure 2A, lane 3). BsrDI REase activity was reconsti- tuted by mixing the cell extracts containing gpORF1 and gpORF2 (data not shown) or by mixing puriﬁed large and small subunits (Figure 2B, lanes 4–8). When 0.8 nM BsrDI