Direct selection of binding proficient/catalytic deficient variants ofBamHI endonuclease

Abstract

Variants of BamHl endonuclease in which the glutamate 1 13 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placing a BamHl recognition sequence, GGATCC, positioned as an operator sequence in an antisense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHl variants. The BamHl endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E.coli carrying the aadA gene construct, and transformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

“…A recent report indicates that EcoRV can bind to DNA in the absence of Ca 2ϩ or Mg 2ϩ under different conditions (44); attempts to visualize a gel shift for PvuII in the absence of metal ions or in the presence of Mg 2ϩ for the catalytically inactive mutants have thus far been unsuccessful. Similar mutagenesis studies have been done on EcoRI (10 -13, 15, 45), EcoRV (18,20,23,24,28), and BamHI (26,27,30). These studies have anticipated or confirmed the predicted roles of residues identified as central from the crystal structures of the respective enzymes.…”

“…There is a relative paucity of mutations described similar to, for example, T37A of EcoRV (28) that are located outside the catalytic domain and bind DNA specifically but are defective in cleavage. In one case where catalytic mutations of BamHI were selected directly using the transcriptional interference method, all the mutations found were in catalytic domain residues (26). This rarity may be due to how the mutations were made and analyzed rather than a reflection of their abundance in nature.…”

“…Binding of Mutants to the PvuII Site-DNA binding was tested with a variation of the transcriptional interference assay (26,39); binding of a catalytically deficient mutant to a PvuII site on a reporter plasmid allowed the expression of spectinomycin resistance (see "Experimental Procedures"). Two mutants gave a significant indication of in vivo binding: D34G and H85A (Table I).…”

“…Structure-function studies have been limited to a small number of type II enzymes. Years of studies have produced an extensive amount of information about EcoRI (10 -18), EcoRV (18 -24), and NaeI (25) and, more recently, BamHI (26,27). These studies have focused on the identification and analysis of residues involved in catalysis, testing alternative mechanisms of catalysis, and understanding how a specific DNA sequence is recognized.…”

“…One explanation for this difficulty is that a change in specificity not only requires recognition of a different DNA sequence, but also requires that the linkage between recognition and catalysis be retained. A class of mutations that might illuminate this linkage is the catalytic mutations, where specific DNA binding is retained but catalysis is reduced (26,30). In particular, mutants in this class that are outside the catalytic center might be deficient in the linkage between recognition and catalysis (28).…”