Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

Abstract

Restriction endonucleases (REases) with 8-base specificity are rare specimens in nature. NotI from Nocardia otitidis-caviarum (recognition sequence 5'-GCGGCCGC-3') has been cloned, thus allowing for mutagenesis and screening for enzymes with altered 8-base recognition and cleavage activity. Variants possessing altered specificity have been isolated by the application of two genetic methods. In step 1, variant E156K was isolated by its ability to induce DNA-damage in an indicator strain expressing M.EagI (to protect 5'-NCGGCCGN-3' sites). In step 2, the E156K allele was mutagenized with the objective of increasing enzyme activity towards the alternative substrate site: 5'-GCTGCCGC-3'. In this procedure, clones of interest were selected by their ability to eliminate a conditionally toxic substrate vector and induce the SOS response. Thus, specific DNA cleavage was linked to cell survival. The secondary substitutions M91V, F157C and V348M were each found to have a positive effect on specific activity when paired with E156K. For example, variant M91V/E156K cleaves 5'-GCTGCCGC-3' with a specific activity of 8.2 x 10(4) U/mg, a 32-fold increase over variant E156K. A comprehensive analysis indicates that the cleavage specificity of M91V/E156K is relaxed to a small set of 8 bp substrates while retaining activity towards the NotI sequence.

Figure 1

Altered cleavage characteristics of NotI variant E156K determined by digestion of plasmid substrate pXba. Lane 1, no enzyme addition (−); Lane 2, digestion with 100 U wt NotI; Lanes 3–9, increasing amounts of variant E156K. Lane M, 1 kb DNA ladder (NEB). All reactions were incubated at 37°C for 60 min in 1× NEB buffer 3.

Figure 2

Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

Figure 3

An illustration of the conditionally toxic selection vector ptoxBAC832 prepared by the MapDraw program of DNASTAR Lasergene. GCT denotes the sequence 5′-GCTGCCGC-3′. GCA denotes the sequence 5′-GCAGCCGC-3′. GCC denotes the sequence 5′-GCCGCCGC-3′. GCGT denotes the sequence 5′-GCGTCCGC-3′.

Figure 4

Evaluation of double variants by digestion of pBR322 (linearized by AflIII). Lane M, 1 kb DNA ladder; Lane 1, incubation with 100 U wt NotI to confirm no cleavage of pBR322; Lane 2, incubation with cell extract from clone EP1 (E156K/V348M); Lane 3, incubation with cell extract from clone EP5 (E156K/F157C); Lane 4, incubation with cell extract from clone EP10 (M91V/E156K); Lane 5, incubation with cell extract containing variant E156K. All reactions were at 37°C for 60 min in 1× NEB buffer 3. Production of 2.9 and 1.5 kb fragments is consistent with cleavage at 5′-TCGGCCGC-3′.

Figure 5

Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × NEB BamHI buffer. (−) indicates no enzyme addition.

Figure 6

Competitive cleavage of two linear substrates by variant E156K/M91V. The 2686 bp substrate was derived from pUC-GCT and the 1778 bp substrate was derived from pUC-NotI. Equimolar amounts of each substrate were mixed and incubated with increasing concentrations of enzyme. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer. (−) indicates no enzyme addition.

Figure 7

Digestion of T7 genomic DNA by variant M91V/E156K. (A) A virtual digest of T7 DNA using NEBcutter (29). Since site 5′-GCGTCCGC-3′ is nicked by this enzyme it was not included in the virtual digest. M is a size marker lane. (B) Actual results of digesting T7 DNA in 1× NEB BamHI buffer for various times at 37°C. A 30-fold molar excess of enzyme was used in each reaction. (C) A control showing no digestion of T7 DNA by 200 U wt NotI.