Cofactor requirements of BamHI mutant endonuclease E77K and its suppressor mutants

Abstract

A mutant BamHI endonuclease, E77K, belongs to a class of catalytic mutants that bind DNA efficiently but cleave DNA at a rate more than 103-fold lower than that of the wild-type enzyme (S. Y. Xu and I. Schildkraut, J. Biol. Chem. 266:4425-4429, 1991). The preferred cofactor for the wild-type BamHI is Mg2+. BamHI is 10-fold less active with Mn2+ as the cofactor. In contrast, the E77K variant displays an increased activity when Mn2+ is substituted for Mg2, in the reaction buffer. Mutations that partially suppress the E77K mutation were isolated by using an Escherirhia coli indicator strain containing the dinD::lacZ fusion. These pseudorevertant endonucleases induce E. coli SOS response (as evidenced by blue colony formation) and thus presumably nick or cleave chromosomal DNA in vivo. Consistent with the in vivo result, the pseudorevertant endonucleases in the crude cell extract display site-specific partial DNA cleavage activity. DNA sequencing revealed two unique suppressing mutations that were 'located within two amino acid residues of the original mutation. Both pseudorevertant proteins were purified and shown to increase specific activity at least 50-fold. Like the wild-type enzyme, both pseudorevertant endonucleases prefer Mg2Y as the cofactor. Thus, the second-site mutation not only restores partial cleavage activity but also suppresses the metai preference a$ well. These results suggest that the Glu-77 residue may play a role in metal ion binding or in enzyme activation (allosteric transition) following sequence-specific recognition.The BamHI restriction endonuclease purified from Bacillus amyloliquefaciens H cleaves the symmetric DNA sequence 5'-GGATCC-3' between the two guanines on both strands (reviewed in reference 17). The coding sequence for BamHI has been cloned and sequenced, and the enzyme has been overexpressed in Escherichia coli (2,3,10). Like all type II restriction endonucleases, BamHI requires the divalent cation Mg2' as a cofactor for cleavage activity. Mn2+ can be substituted for Mg2+ in the reaction, but the enzyme activity is reduced (24). Mn2+ also decreases the enzyme specificity, allowing BamHI to cleave noncanonical sequences (BamHI star sites; 10, 14). The DNA-binding activity, however, is independent of metal cofactor (17, 28).The SOS regulon consisting of about 20 genes in E. coli is negatively regulated by the LexA repressor (reviewed in references 19, 22, and 25). The repressor can be cleaved and inactivated by the RecA protein to derepress the genes in the regulon. The proteolytic activity of the RecA protein is activated by a signal (e.g., single-stranded DNA or oligonucleotides) upon DNA damage or inhibition of DNA replication. As the result of DNA damage or DNA replication interference, the genes in the SOS regulon are expressed at an increased level. E. coli indicator strain AP1-200 contains a dinD: :lacZ fusion in which the lactose operon is fused to a DNA damage-inducible promoter (12). --Galactosidase expression is increased upon treatment of the cells with UV, mitomycin...

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.…”

“…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.…”

Alternative Roles for Metal Ions in Enzyme Catalysis and the Implications for Ribozyme Chemistry

“…146 The activity of Glu-77/Lys mutants is enhanced in the presence of suppressor mutations that may optimize the positioning of the mutant cationic side chain. 147 Thus, in both studies 144,145 the term "two-metal ion mechanism" is used even though EcoRV contains only one metal ion (although replacement of the ammonium side chain of Lys-92 by Glu-92/Mn 2+ restores activity), 144 while BamH1 contains two metal ions 145 (one of which may be replaced by the ammonium side chain of Lys). 146,147 These results indicate that metal ions and positively charged amino acid side chains can be interchangeable for classes of enzyme that share a common mechanism.…”

Nonhomologous End Joining during Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae

“…In principle, it is possible that the BamHI enzyme creates a double-strand break to mediate integration events or that it binds to BamHI recognition sites to bring the recombination partners together. Furthermore, since restriction enzymes show nonspecific weak DNA binding (46), restriction enzymes could possibly bind to the transforming DNA and enhance uptake of such DNA indirectly, leading to an increase …”

“…To address this possibility, we used purified BamHI-E77K protein, which binds to the BamHI site but cleaves DNA at a rate 1,000-fold lower than that of wild-type enzyme (46). The BamHI-E77K protein did not increase the efficiency of integration ( Table 1), indicating that the cutting activity of the restriction enzyme is necessary to mediate REMI events.…”