Useful tool to generate unidirectional deletion vectors by utilizing the star activity of BamHI in an NcoI-BamHI-XhoI cassette

It has been shown that restriction endonucleases can cleave DNA sequences that are similar but not identical to their cognate recognition sequences under some nonstandard conditions (1). This phenomenon is mediated by the so-called “star” activity of restriction endonucleases. In general, biologists try to avoid star activity when performing restriction endonuclease digests in order to achieve specific cleavage of DNAs. However, we have found a way to take advantage of this adverse property of restriction endonucleases to facilitate molecular cloning. In this study, we developed a rapid, convenient, economical, and versatile method (diagrammed in Figure 1A) to facilitate large-scale molecular cloning and functional analyses of gene products.

In this report, we use the human ABCB5 gene, a novel ATP-binding cassette (ABC) transporter, as our target gene. The ABCB5-specific primers used were the ABCB5 forward primer (5′-AAGGATCCATTGCTTCTCGGCCTTTTGGCTAAG-3′) and the reverse primer (5′-AACTCGAGTCAC-TGCACTGACTGTGCATTCACT-3′) (K.G. Chen and M.M. Gottesman, unpublished data). The boldfaced and underlined bases represent the restriction sites BamHI and XhoI, which we used to facilitate subcloning and exploit the star activity of BamHI. All restriction endonucleases used in this study were purchased from New England Biolabs (Beverly, MA, USA). A conventional reverse transcription PCR (RT-PCR) protocol was used to amplify the ABCB5 cDNA sequences. The total cDNAs were subjected to denaturation at 94°C for 30 s, followed by 35 cycles of amplification for 30 s at 94°C, 30 s at 55°C, and 3 min at 72°C.

In order to generate unidirectional star activity of an enzyme in an expression cassette, the PCR fragments were doubly digested by both BamHI and XhoI in the presence of SuRE/Cut Buffer B (10 mM Tris-HCl, 5 mM MgCl₂, 100 mM NaCl, 1 mM 2-mercaptoethanol, pH 8.0, at 37°C) from Roche Applied Science (Indianapolis, IN, USA) and then subcloned into the BamHI and XhoI sites of the pTM1 vector, a vaccinia virus-based expression vector that has been extensively used in our laboratory for membrane protein expression (2). To generate serial deletion constructs for pTM1 so that the first ATG site of ABCB5 for the protein translation was placed closely downstream from the NcoI site to achieve high transcriptional efficiency (2,3), we further doubly digested the pTM1-ABCB5 (NcoI-BamHI-XhoI) vector by NcoI-BamHI in the presence of both NEBuffer 2 [10 mM Tris-HCl, 10 mM MgCl₂, 50 mM NaCl, 1 mM dithiothreitol (DTT), pH 7.9] and bovine serum albumin (BSA; 100 µg/mL) at 37°C for 2 h or 24 h, which is likely to produce a digestion pattern caused by the star activity of the enzymes. The digested sample was subjected to electrophoresis on a 2% agarose gel, and the desired fragments were excised, purified by the GENECLEAN® II Kit (Qbiogene, Carlsbad, CA, USA), and directly ligated by DNA T4 ligase (Roche Applied Science) at 16°C overnight. Two microliters of the ligates were used to transform the Escherichia coli (DH5α) strain (Invitrogen, La Jolla, CA, USA). The plasmids with altered sizes were isolated and sequenced using the sequencing core facility at the National Cancer Institute (Bethesda, MD, USA).

The ABCB5 PCR fragments (approximately 2.9 kb, Figure 1B) were obtained by PCR and subcloned into the BamHI and XhoI sites of the pTM1 vector (Figure 2A) to obtain pTM1-ABCB5 (NcoI-BamI-XhoI) (Figure 2B). We further doubly digested the pTM1-ABCB5 (NcoI-BamHI-XhoI) vector by NcoI-BamHI, which produced a digestion pattern caused by the star activity of the enzymes (Figure 1C). The plasmids with altered sizes were identified (Figure 1D). Of these, the majority of clones contained an intact backbone of the pTM1 vector, as indicated by a 5.4-kb band. The length of the inserts (the ABCB5 fragments) identifies the fragments that were cleaved by BamHI or BamHI* (BamHI star activity). For example, one clone contained a 2.4-kb insert that was the result of a deletion of a large fragment of ABCB5 (Figure 1D, clone 20). This has been confirmed by sequencing (Figure 2C).

It is believed that any restriction endonuclease can be made to cleave noncanonical sites under certain altered conditions (such as changes in pH or ionic strength; References 4–6). New England Biolabs has confirmed that many restriction endonucleases exhibit star activity. We have noted that star activity was correlated with the length of the enzyme digestion (more than 2 h) in a water bath due to an altered ionic strength resulting from the evaporation of water from the reaction mixture in the Eppendorf® tube, which condenses at the top of the tube. The most common types of altered activity can result in single base substitutions, disruption of the outer bases in a cognate sequence, and single-strand nicking (4). Previous studies showed that EcoRI* cut the sequence NAATTN or any sites that differ in a single base change under certain conditions, such as an elevated pH and low ionic strength (5,6). We designed an NcoI-BamHI-XhoI cassette in the pTM1 vector, in which our gene of interest was inserted into the BamHI-XhoI sites, resulting in the NcoI-BamHI-ABCB5 (2.9 kb)-XhoI cassette (Figure 2B). The star activity of NcoI was limited in this NcoI-BamHI combination after the linearization of the plasmids. Moreover, the star activities of NcoI and BamHI are mutually exclusive in this combined digest, which allows for unidirectional generation of deletions (Figure 1D, clones 20 and 17). It allows BamHI to exert its star activity in a unidirectional manner, whereas NcoI* might produce a nick in a DNA strand (Figure 2C). Thus, BamHI, which recognizes and cleaves the motif sequence GGATCC, displayed a new phenomenon that involves the relaxation of sequence specificity and cleaves a noncognate site, TGATCC, under a prolonged reaction in the presence of NcoI. In this study, we chose BamHI* simply as one example to demonstrate the feasibility of our proposed application. The restriction endonucleases from New England Biolabs that have been demonstrated to exhibit star activity in the cassette shown in Figure 2A include BamHI, EcoRI, PstI, and SalI. Other restriction endonucleases include ApoI, AseI, BssHII, EcoRV, HindIII, HinfI, PvuII, ScaI, TaqI, and XmnI (see New England Biolabs, http://www.neb.com/neb). Thus, the redundancy of the above restriction endonucleases with star activity would allow us to choose different enzyme combinations for a given gene sequence to generate the desired constructs. Our procedures for generating a cluster of constructs can be completed in approximately 4 days, compared to other conventional techniques, which usually take about 1 week to produce the desired constructs. Clearly, this technique provides a rapid, convenient, and economical way to generate unidirectional deletions.