Binding kinetics and footprinting of TaqI endonuclease: effects of metal cofactors on sequence-specific interactions.
Restriction endonucleases achieve sequence-specific recognition and strand cleavage through the interplay of base, phosphate backbone, and metal cofactor interactions. In this study, we investigate the binding kinetics of TaqI endonuclease using the wild-type enzyme and a binding proficient, catalysis deficient mutant TaqI-D137A both in the absence of a metal cofactor and in the presence of Mg2+ or Ca2+. As demonstrated by gel mobility shift analyses, TaqI endonuclease requires a metal cofactor for achieving high-affinity specific binding to its cognate sequence, TCGA. In the absence of a metal cofactor, the enzyme binds all DNA sequences (TaqI cognate site, star site, and nonspecific site) with essentially equal affinity, thereby exhibiting little discrimination. The dissociation constant of the cognate sequence in the presence of Mg2+ at 60 degrees C is 0. 26 nM, a value comparable to our previously reported Km of 0.5 nM measured under steady-state conditions. The TaqI-TCGA-Mg2+ complex is stable, with a half-life of 21 min at 60 degrees C. The boundary of the protein-DNA interface is approximated to be about 18 bp as determined by DNase I footprinting. Data from this study support the notion that a metal cofactor plays a critical role for achieving sequence-specific discrimination in a subset of nucleases, including TaqI, EcoRV, and others.
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Department of Microbiology and Immunology, Strang Cancer Prevention Center, The Joan and Sanford I. Weill Medical College of Cornell University, New York 10021, USA. wgc@mail.med.cornell.edu
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry
D002413:Cations, Divalent; D009838:Oligodeoxyribonucleotides; D003850:Deoxyribonuclease I; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases; D008274:Magnesium; D002118:Calcium