ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF THE BAMHI RESTRICTION-ENDONUCLEASE

Two heat-sensitive R . BamHI mutants, T157I and P173L, and one cold-se nsitive R . BamHI mutant, T114I, were isolated after chemical mutagene sis of the bnmhIR gene that codes for the restriction endonuclease Bnm HI (R . BnmHI). The thermosensitivity of T114I, T157I and P173L is rev ealed by the 10(2)-10(3) lower plating efficiency at the non-permissiv e temperature of strains bearing these alleles. The conditional-lethal phenotype can be rescued by introduction of the cognate bnmhlM gene i nto the same cell. The mutant enzymes induce the SOS response in vivo and display reduced phage restriction activity. The P173L protein, whe n expressed at 30 degrees C and purified, shows reduced thermostabilit y at 65 degrees C. T157I and P173L mutants yield different intermediat es during partial trypsin digestion. The conditional-lethal BnmHI muta nts could be used to deliver in vivo DNA cleavage and for further isol ation of relaxed-specificity mutants.