1/3/2024 0 Comments Noti siteOther progress has been made with altering the specificity of the homing endonuclease I-CreI ( 8). Challenging the mutant library in vitro with GsuI (5′-CTG GAG-3′) enabled isolation of an Eco57I variant with methylation specificity for 5′-CTG RAG-3′ and corresponding endonuclease activity upon restoration of the catalytic residue by site-directed mutagenesis. A cleavage minus variant was randomly mutated and subjected to the methylase selection procedure. This study of BsoBI is the first example where a DNA-damage indicator strain was used for the primary purpose of increasing REase specific activity.Ī novel approach was applied to Eco57I (5′-CTG AAG-3′) to obtain a Type IIG enzyme with altered specificity ( 6, 7). Allele D246A was then subjected to random mutagenesis and in vivo screening to isolate five unique variants with increased activity. In a structure-guided study of BsoBI (specificity = 5′-CYCGRG-3′), variant D246A was found to display a preference for cleavage of 5′-CCCGGG-3′ ( 5). Remarkably, the two residues were K133 and S172. In subsequent X-ray structural studies of wt BstYI, only two amino acid residues were found making base-specific contacts to the 5′-AGATCT-3′ sequence ( 4). The variant K133N/S172N possessed a 12-fold preference for AGATCT over AGATCC or GGATCT and cleavage of GGATCC was no longer detectable. The BstYI evolution procedure yielded a variant with a marked increase in substrate specificity ( 3). More recently, a genetic selection and screening procedure lead to the discovery of two important side chains for BstYI specificity (wt specificity is 5′-RGATCY-3′ where R = A or G, Y = C or T). Many of the efforts have employed a DNA-damage indicator strain as pioneered by Heitman and Model ( 1, 2) who developed an SOS induction assay to screen for relaxed variants of EcoRI in order to identify amino acids that make specific contacts to cognate DNA. 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.Ĭonsiderable progress has been made in isolating restriction endonucleases (REases) with altered substrate specificity. For example, variant M91V/E156K cleaves 5′-GCTGCCGC-3′ with a specific activity of 8.2 × 10 4 U/mg, a 32-fold increase over variant E156K. The secondary substitutions M91V, F157C and V348M were each found to have a positive effect on specific activity when paired with E156K. Thus, specific DNA cleavage was linked to cell survival. In this procedure, clones of interest were selected by their ability to eliminate a conditionally toxic substrate vector and induce the SOS response. In step 2, the E156K allele was mutagenized with the objective of increasing enzyme activity towards the alternative substrate site: 5′-GCTGCCGC-3′. 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). Variants possessing altered specificity have been isolated by the application of two genetic methods. 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. Restriction endonucleases (REases) with 8-base specificity are rare specimens in nature.
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