title: The domain organization of NaeI endonuclease: separation of binding and catalysis.
abstract: NaeI is a remarkable type II restriction endonuclease. It must bind two recognition sequences to cleave DNA, forms a covalent protein-DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity. The latter activities apparently derive from reactivation of a cryptic DNA ligase active site. Here, we demonstrate that NaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa. The domains were purified by cloning. The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intact NaeI. Analytical ultracentrifugation showed this domain to be a monomer in solution. The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association. DNA greatly inhibited proteolysis of the linker region. The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure for NaeI.
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Pathology, University of North Carolina Medical School, Chapel Hill, NC 27599-7295, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Colandene
authors.forename: M D
authors.initials: MD
authors.lastname: Topal
mesh_terms: D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D016296:Mutagenesis; D011487:Protein Conformation; D015202:Protein Engineering; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific