Pyrophosphorolytic dismutation of oligodeoxynucleotides by terminal deoxynucleotidyltransferase

Terminal transferase (TdT), when incubated with a purified P-32-5'-end-labe led oligonucleotide of defined length in the presence of Co2+, Mn2+ or Mg2 and 2-mercaptoethanol in cacodylate or HEPES buffer, pH 7.2, exhibits the ability to remove a 3'-nucleotide from one oligonucleotide and add it to th e 3'-end of another. When analyzed by urea-PAGE, this activity is observed as a disproportionation of the starting oligonucleotide into a ladder of sh orter and longer oligonucleotides distributed around the starting material, Optimal metal ion concentration is 1-2 mM, All three metal ions support th is activity with Co2+, Mn2+ congruent to Mg2+, Oligonucleotides p(dT) and p (dA) are more efficient substrates than p(dG) and p(dC) because the latter may form secondary structures, The dismutase activity is significant even i n the presence of dNTP concentrations comparable to those that exist in the nucleus during the G(1) phase of the cell cycle. Using BetaScope image ana lysis the rate of pyrophosphorolytic dismutase activity was found to be onl y moderately slower than the polymerization activity, These results may hel p explain the GC-richness of immunoglobulin gene segment joins (N regions) and the loss of bases that occur during gene rearrangements in pre-B and pr e-T cells.