Figure 4
Conserved region in the HsdR polypeptide
of restriction enzymes EcoKI, EcoAI and EcoR124I.

In order to remove any possibility of DNA cleavage by EcoR124I, we will use site-directed mutagenesis to destroy the motif involved in DNA cleavage. The involvement of motif X in endonuclease activity was demonstrated for EcoAI (Janscak et al., 1999a) and EcoKI (Davies et al., 1999) . In both cases single amino acid substitutions in the conserved residues (D,E,K) of the D-X13-14-E-X-K sequence motif X (Figure 4) uncoupled the DNA translocation and DNA cleavage activities of the enzymes. Therefore, using the great experience of these techniques available at the Institute of Microbiology, Prague, we will concentrate on mutagenesis of the three-conserved amino acid residues, D151, E165 and K167 in the endonuclease motif of HsdR subunit of restriction enzyme EcoR124I. The origin repair method (Ohmori, 1994) will be employed for site-directed mutagenesis. The restriction phenotype of mutant endonucleases will be determined in vivo. For in vitro analysis, the mutant HsdR subunits, will be separately overproduced, purified and mixed with purified methylase to reconstitute the endonuclease. The ATPase activity of EcoR124I mutants will be measured by a colorimetric estimation of the inorganic phosphate released by ATP hydrolysis.

Outcomes:

All cleavage mutants of HsdR subunit were purified and mutant endonucleases reconstituted from the wt methylase and individual mutant HsdR subunits were analysed in vitro. Cleavage assay proved in agreement with the in vivo phenotype that none of the mutant enzyme is able to cleave the plasmid DNA to its linear form. DNA binding, ATPase and translocation activities are similar to the wt enzyme.