Initiator role of double stranded DNA in terminal transferase catalyzed polymerization reactions

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Abstract
Binding of the 58 kDa monomer and 44 kDa αβ dimer forms of terminal deoxynucleotidyl transferase to double stranded DNA was, demonstrated by gel retardation and tiyptophan fluorescence quenching. The dissociation constants and cooperativity parameters were similar to those that have been determined for binding of these two forms of terminal transferase to single stranded DNA. However, the double stranded DNA binding site size of 10 nucleotides was half the size expected. The efficacy of blunt ended DNA as an initiator in the polymerization reaction catalyzed by terminal transferase was demonstrated by radiometric assays and product analyses on ajarose gels. The initial reaction kinetics indicated that dGTP but not dATP was added efficiently to a blunt double stranded DNA 3′ end. These results are correlated with current models for in vivo terminal transferase function.