Random Gene Dissection: A Tool for the Investigation of Protein Structural Organization

Abstract

To investigate the domain structure of proteins and the function of individual domains, proteins are usually subjected to limited proteolysis, followed by isolation of protein fragments and determination of their functions. We have developed an approach we call random gene dissection (RGD) for the identification of functional protein domains and their interdomain regions as well as their in vivo complementing fragments. The approach was tested on a two-domain protein, the type IIS restriction endonuclease BfiI. The collection of BfiI insertional mutants was screened for those that are endonucleolytically active and thus induce the SOS DNA repair response. Sixteen isolated mutants of the wild-type specificity contained insertions that were dispersed in a relatively large region of the target recognition domain. They split the gene into two complementing parts that separately were unable to induce the SOS DNA repair response. In contrast, all 19 mutants of relaxed specificity contained the cassette inserted into a very narrow interdomain region that connects BfiI domains responsible for DNA recognition and for cleavage. As expected, only the N-terminal fragment of BfiI was required to induce SOS response. Our results demonstrate that RGD can be used as a general method to identify complementing fragments and functional domains in enzymes.

Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants

“…The N-terminus of BfiI was identified as a non-specific nuclease based on biochemical, genetic, and structural studies [21][22][23]. Based on the BfiI/BmrI amino acid sequence alignment, two versions of the N-terminal domains, BmrI192 (residues 1 to 192), and BmrI200 (residues 1-200, residue Thr200 substituted for Cys) were independently expressed in E. coli.…”