Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase.
NaeI is a type IIe endonuclease that interacts with two DNA recognition sequences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structures parallel the two functionalities recognized in NaeI, endonuclease and topoisomerase. In this study, we report evidence for mutations that break interdomain functional communication in a NaeI-DNA complex. Deletion of the initial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domain. 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 flanking the NaeI recognition sequence. Substituting Ala for Arg182 within this region had no apparent effect on DNA binding but greatly reduced the extent 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-fold. Substituting Ala for Lys189 altered flanking sequence recognition. Residues 182-192 are away from the Endo domain responsible for cleavage and also face away from the modeled DNA binding faces of the apoprotein crystal structure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.
Colandene
J D
JD
Lineberger Comprehensive Cancer Center, University of North Carolina Medical School, Chapel Hill, North Carolina 27599-7295, USA.
Topal
M D
MD
D000409:Alanine / Q000235:genetics; D001120:Arginine / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D058619:Maltose-Binding Proteins; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001120:Arginine; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific; D004264:DNA Topoisomerases, Type I; D000409:Alanine