Abstract
Derivation of the bamhIR sequence (Brooks, J. E., Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Ives, C. C., Mazzola, L. M., Slatko, B. E., and Benner, J. S. (1991) Nucleic Acids Res., in press), the gene coding for BamHI endonuclease, has facilitated construction of an Escherichia coli strain that overproduces BamHI endonuclease (W. E. Jack, L. Greenough, L. F. Dorner, S. Y. Xu, T. Strezelecka, A. K. Aggarwal, and I. Schildkraut, submitted for publication). As expected, low-level constitutive expression of the bamhIR gene in E. coli from the Ptac promotor construct is lethal to the host unless the bamHIM gene, which encodes the BamHI methylase, is also expressed within the cell. We identified four classes of BamHI endonuclease variants deficient in catalysis by selecting for survival of a host deficient for bamHIM gene, transformed with mutagenized copies of the bamhIR gene, and then screening the surviving cell extracts for DNA cleavage and binding activities. Class I variants (G56S, G91S/T153I, T114I, G130R, E135K, T153I, T157I, G194D) displayed 0.1-1% of the wild-type cleavage activity; class II variant (D94N) lacked cleavage activity but retained wild-type DNA binding specificity; class III variants (E77K, E113K) lacked cleavage activity but bound DNA more tightly; class IV variants (G56D, G90D, G91S, R122H, R155H) lacked both binding and cleavage activities. Variants with residual cleavage activities induced the E. coli SOS response and thus are presumed to cleave chromosomal DNA in vivo. We conclude that Glu77, Asp94, and Glu113 residues are essential for BamHI catalytic function.
Highlights
S . (1991)Nucleic Acids Res., in press), the residues that are critical for the catalytic function
W-leveclonstitutiveexpression of the burnhIR gene in E. coli from the Pta, promotor construct is lethal to the host unless the bumHIM gene, which encodes the BamHImethylase, is expressed within the cell
Mg2+is required as acofactor for type I1 restriction enzyme cleavage activity
Summary
’$ To whom correspondence should be addressed. The abbreviations used are: bp, base pair(s); PAGE, polyacrylamide gel electrophoresis. Plasmid DNA was used as a template for dideoxy termination w,*,r:,, sequencing (13). Isopro- variants,undiluted or diluted in sonication huffer, were incuhated pylthiogalactoside was added to0.4 mM and incuhation was continued with 1 pg ofA-DNA a t 37 "C for 30 min in 20 pl Reactions in 1 ml of sonication huffer Cell dehris wasremoved by centrifugation and the supernatantwas diluted 2-fold hy addition of mM EDTA, 2% NaDodSO,, 0.1% hromphenol blue). DNA hands were resolved hy agarose gel electrophoresis. Electrophoresis of DNA-RamHI complex in native polyacrylamide gel was performed as descrihed (15). I O nM of end-labeled DNA was incuhated with various dilutions of crude cell
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