Target recognition by EcoKI: The recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity

We report a genetic and biochemical analysis of a target recognition domain (TRD) of EcoKI, a type I restriction and modification enzyme. The TRDs of type I R-M systems are within the specificity subunit (HsdS) and HsdS confe rs sequence specificity to a complex endowed with both restriction and modi fication activities. Random mutagenesis has revealed that most substitution s within the amino TRD of EcoKI, a region comprising 157 amino acid residue s, have no detectable effect on the phenotype of the bacterium, even when t he substitutions are nonconservative. The structure of the TRD appears to b e robust. All but one of the six substitutions that confer a restriction-de ficient, modification-deficient (r(-)m(-)) phenotype were found to be in th e interval between residues 80 and 110, a region predicted by sequence comp arisons to form part of the protein-DNA interface. Additional site-directed mutations affecting this interval commonly impair both restriction and mod ification. However, we show that an r(-) phenotype cannot be taken as evide nce that the EcoKI complex lacks endonuclease activity; in response to even a slightly impaired modification efficiency, the endonuclease activity of EcoKI is destroyed by a process dependent upon the ClpXP protease. Enzymes from mutants with an r(-)m(-) phenotype commonly retain some sequen ce-specific activity; methylase activity can be detected on hemimethylated DNA substrates and residual endonuclease activity is implied whenever the v iability of the r(-)m(-) bacterium is dependent on ClpXP. Conversely, the v iability of ClpX(-) r(-)m(-) bacteria can be used as evidence for little, o r no, endonuclease activity. Of 14 mutants with an r(-)m(-) phenotype, only six are viable in the absence of ClpXP. The significance of four of the si x residues (G91, G105, F107 and G141) is enhanced by the finding that even conservative substitutions for these residues impair modification, thereby conferring an r(-)m(-) phenotype. (C) 2001 Academic Press.