Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymic activity, not, however, the preference of this restriction endonuclease for cleavage within the site -GAATTC-

Citations Over TimeTop 21% of 1989 papers

Abstract

According to the X-ray structure analysis of an EcoRI-oligodeoxynucleotide complex [McClarin et al. (1986) Science 234, 1526], sequence specificity is mediated by 12 hydrogen bonds, 6 from each of the two identical subunits of the dimeric enzyme to the recognition site -GAATTC-: Arg200 forms two hydrogen bonds with guanine, while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. Changing the hydrogen-bonding potential at the recognition site without perturbing the rest of the interface should lead to the recognition of degenerate sequences [Rosenberg et al. (1987) in Protein Engineering (Oxender, D. L., & Fox, C. F., Eds.) pp 237-250, Liss, New York]. We have shown previously that replacing Glu144 by Gln and Arg145 by Lys affects the activity of the enzyme, not, however, its specificity [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. We show now that also the mutation of Arg200 to Lys, the double mutation Glu144Arg145 to GlnLys, and the triple mutation Glu144Arg145Arg200 to GlnLysLys do not lead to a detectable degeneracy of the specificity of cleavage by EcoRI but significantly impair the catalytic activity of this enzyme. A detailed analysis of the steady-state kinetics of cleavage of pUC8 DNA and a tridecadeoxynucleotide substrate demonstrates that the reduction in activity for all DNA binding site mutants investigated so far is mainly due to a decrease in kcat, with the exception of the Arg200 to Lys mutant, which is only impaired in its KM.(ABSTRACT TRUNCATED AT 250 WORDS)

Related Papers

• Restriction enzyme analysis of bovine herpesvirus-1 DNA from an Indian isolate.(1993)
• → Physical Maps for HSV Type 2 DNA with Five Restriction Endonucleases(1978)50 cited
• Restriction endonuclease analysis of DNA from Indian isolates of bovine herpesvirus 1.(1997)
• → The molecular weight of the egg drop syndrome (EDS) avian adenovirus (strain B8/78) DNA estimated by digestion with restriction endonuclease enzyme R�EcoRI(1980)7 cited
• Restriction Endonuclease Analysis of Egg Drop Syndrome Virus Isolated from the West Region of Henan Province(2003)