title: Comparison of the two murine terminal [corrected] deoxynucleotidyltransferase terminal isoforms. A 20-amino acid insertion in the highly conserved carboxyl-terminal region modifies the thermosensitivity but not the catalytic activity.
abstract: Terminal deoxynucleotidyltransferase (TdT) catalyzes the addition of nucleotides to 3'-hydroxyl ends of DNA strands in a template-independent manner and has been shown to add N-regions to gene segment junctions during V(D)J recombination. TdT is highly conserved in all vertebrate species, with a second isoform, characterized by a 20-amino acid insertion near the COOH-terminal end, described only in the mouse. The two murine isoforms differ in their subcellular localization, and the long isoform (TdTL) has previously been found to be unable to add N-regions. Using purified protein produced in a high level expression system in Escherichia coli, we were able to carry out detailed catalytic comparisons of these two TdT isoforms. We discovered that TdTL exhibits terminal transferase activity with kinetic parameters similar to those of the conserved TdT isoform (TdTS). We observed, however, that TdTL is inactivated at physiologic temperature but stable at lower temperatures. This thermal sensitivity of TdTL polymerase activity is not correlated with a significant change in the circular dichroism spectrum of the protein. Thus, the 20-amino acid insertion in TdTL does not affect the catalytic activity but modifies the thermosensitivity.
authors.affiliation: Unité de Génétique et Biochimie du Développement, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.
authors.forename: J B
authors.initials: JB
authors.lastname: Boulé
authors.forename: F
authors.initials: F
authors.lastname: Rougeon
authors.forename: C
authors.initials: C
authors.lastname: Papanicolaou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D017124:Conserved Sequence; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D007527:Isoenzymes; D004253:DNA Nucleotidylexotransferase