Interaction of terminal transferase with single-stranded DNA.
A 58-kDa monomer of terminal transferase was isolated from calf thymus using a monoclonal antibody affinity column. The enzymatic activity was comparable to that of the 44-kDa alpha beta dimer isolated by conventional methods. Binding of the two enzyme forms to single-stranded DNA was monitored by fluorescence. The site size of both forms was approximately 11 +/- 2 nucleotides. Binding of the 44-kDa alpha beta dimer to polydeoxyadenosine was examined under several conditions. The cooperativity parameter increased from about 90 in the presence of Mg2+ to 300-400 in the absence of Mg2+. The observed dissociation constant of 3-5 microM was essentially independent of salt concentration, whereas the intrinsic dissociation constant decreased about 5-fold in the presence of Mg2+. The binding parameters of the 58-kDa monomer were independent of buffer composition and were similar to those of the 44-kDa alpha beta dimer in the presence of Mg2+. These results indicate that the additional 14-kDa peptide sequences present in the high molecular mass monomer form are not part of the DNA-binding site of terminal transferase.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004253:DNA Nucleotidylexotransferase / Q000302:isolation & purification / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013950:Thymus Gland / Q000201:enzymology*
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase