On the repair of DNA breaks and the specificity of the EcoRI restriction enzyme

We took a genetic approach to study the well characterized EcoRI restriction enzyme, an endonuclease that cleaves DNA molecules at the sequence GAATTC. First, a series of temperature-sensitive EcoRI endonuclease alleles were isolated. Temperature shifts with these alleles revealed that in vivo DNA scission induces the E. coli SOS DNA repair response. However, neither SOS induction nor recombination are required to repair these lesions. DNA ligase is required and may suffice to repair EcoRI breaks in the E. coli chromosome.; To monitor DNA scission in vivo we employed strains carrying the lactose operon fused to an SOS inducible promoter. After DNA scission, these strains produce {dollar}beta{dollar}-galactosidase and form blue colonies on X-Gal medium. Using this assay, two approaches were taken to isolate EcoRI mutants altered or disrupted in substrate specificity. First, amino acids (E144, R145, R200) implicated in substrate binding by the crystal structure were subjected to site-directed mutagenesis. Of 50 of the 60 possible substitutions, several alleles retain weak endonuclease activity which is of wild-type specificity. Therefore the simple hydrogen bond model proposed from the crystal structure is insufficient to explain substrate recognition and additional interactions must participate in the substrate-enzyme complex.; In the second approach, mutants of an EcoRI TS allele were isolated which conditionally induce the SOS response in spite of the protective methylase. These mutant proteins exhibit enhanced cleavage activity at EcoRI{dollar}sp{lcub}*{rcub}{dollar} sites. Four of five isolated mutations lie at the DNA-protein interface and may directly alter or disrupt substrate recognition. One other (H114Y) lies far from the binding or cleavage sites and falls three amino acids away from a previously described mutation, E111G (King, et al., 1986, 1988), which severely impairs DNA cleavage without altering DNA binding. These mutations support a model whereby DNA scission by the EcoRI endonuclease is allosterically activated upon substrate binding: the E111G mutation may inhibit this conformational change while the H114Y mutation may render it more facile such that additional DNA sequences act as allosteric effectors and trigger cleavage.