Human proteins controlling cellular development, gene regulation, and DNA recombination, do so by recognizing specific sequences of DNA. A central question in biology is how proteins recognize DNA in a sequence-specific manner. In this context, TaqI endonuclease has served as a model system by virtue of its million fold specificity for its cognate sequence, T CGA, its ease of production and assay, and its thermophilicity. The present application aims to expand our understanding of substrate and co- factor determinants of specific binding and cleavage by TaqI endonuclease. To characterize the interactions between TaqI and the phosphate backbone, a series of site/stereo-specific methylated substrates will be synthesized and kinetic parameters (k/st and K/m) determined. The hypothesis and DNA distortion plays and important role in sequence specific recognition, will be tested using """"""""circular doughnut"""""""" and other new substrates. Photo-affinity and chemical- crosslinking agents used under sequence-specific binding conditions may help identify important amino acid residues involved in binding and cleavage. This will be complemented by genetic analysis of """"""""null"""""""" mutants and isoschizomers such as Tth HB8I endonuclease. Ultimately, these results will be correlated with structural determinations on the TaqI-DNA complex using X-ray crystallography and NMR spectroscopy. In addition to serving as a valuable model for sequence-specific recognition, the exquisite specificity of TaqI endonuclease is being employed in the field of DNA diagnostic. This laboratory has developed a procedure using thermophilic restriction enzymes, such as TaqI, to detect 94% of single-base mutations in a given cancer gene. This method, which combines the best properties of the polymerase chain reaction (PCR) and the ligase chain reaction (LCR, developed in this laboratory) will be able to detect cancer-causing mutations at the earliest stage, when the mutation is present in only 1 out of 10(6) normal cells. Unlike the enzymes Taq polymerase and Taq ligase which survive thermo-cycling, TaqI is only thermophilic. This proposal has developed systematic strategies for rendering TaqI thermostable. These include generating TaqI-TthHB8I chimeras, tethering two subunits to make a """"""""Gemini dimer"""""""", and/or introducing additional mutations in the variable regions of the enzyme. This approach may yield important insights into the elements which contribute to protein thermostability. The effectiveness of new therapies for hereditary and infectious diseases will be dependent on the molecular information derived from these diagnostic tests. Since these tests employ thermostable DNA recognition proteins (Taq polymerase, Taq ligase, and thermostable TaqI), a deeper understanding of their mechanism of action is necessary step for the advancement of molecular medicine.
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