Biochemistry of terminal deoxynucleotidyl transferase. Conditions for and characterization of ultraviolet light mediated substrate cross-linking to terminal deoxynucleotidyl transferase.

Calf thymus terminal deoxynucleotidyl transferase may be cross-linked to substrate deoxynucleoside triphosphate as well as ATP in a reaction mediated by ultraviolet irradiation. The cross-linking requires the presence of divalent cation but is independent of primer. The cross-linking of substrate to enzyme is extremely sensitive to substrate binding site directed reagents, e.g. pyrophosphate and pyridoxal 5'-phosphate, and to o-phenanthroline, a zinc-directed chelator. The cross-linking reaction is competitively inhibited by all four deoxynucleoside triphosphates, ATP, and GTP, but is resistant to sulfhydryl reagents. When primer is provided in the reaction mixture, cross-linking of both substrate and primer to 26K subunit of terminal deoxynucleotidyl transferase may be demonstrated.