Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase

NaeI is a type lie endonuclease that interacts with two DNA recognition seq uences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structu res parallel the two functionalities recognized in NaeI, endonuclease and t opoisomerase. In this study, we report evidence for mutations that break in terdomain functional communication in a NaeI-DNA complex. Deletion of the i nitial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domai n. Deletions within a small region of the C-terminal DNA binding domain of NaeI (amino acids 182-192) altered the recognition by NaeI of sequences fla nking the NaeI recognition sequence. Substituting Ala for Arg182 within thi s region had no apparent effect on DNA binding but greatly reduced the exte nt of DNA cleavage even though it is not part of the catalytic Endo domain. Substituting Ala for Ile185 reduced the extent of DNA binding about 1000-f old. Substituting Ala for Lys189 altered flanking sequence recognition. Res idues 182-192 are away from the Endo domain responsible for cleavage and al so face away from the modeled DNA binding faces of the apoprotein crystal s tructure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.