He has used TdT as a model DNA polymerase to analyze the process of substrate and primer binding. Several site-specific reagents and procedures for the site-specific covalent modifications have been developed to label the desired site(s) in TdT. Using an affinity labeling protocol, we have demonstrated the identity of the substrate binding domain in the 3 molecular weight species of TdT. He will now define the primary amino acid sequence of that domain as well as identify the amino acid residue which participates in binding of the substrate triphosphate. Studies are also planned to define the primer binding site domain in TdT which utilizes a) covalent crosslinking of defined length oligomeric DNA primer to various MW species of TdT and b) covalent chemical linkage of Ap5A, a newly found inhibitor, to the primer binding site. An analysis of peptides involved in primer binding and their primary amino acid sequence together with investigation on the mechanism of inhibition of some Ap5A analogues is expected to provide a general geometry of the active site structure involved in substrate and primer binding in TdT.
