Pyrophosphorolytic dismutation of oligodeoxy-nucleotides by terminal deoxynucleotidyltransferase.
Terminal transferase (TdT), when incubated with a purified(32)P-5"-end-labeled oligonucleotide of defined length in the presence of Co(2+), Mn(2+)or Mg(2+)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 the 3"-end of another. When analyzed by urea-PAGE, this activity is observed as a disproportionation of the starting oligonucleotide into a ladder of shorter and longer oligonucleotides distributed around the starting material. Optimal metal ion concentration is 1-2 mM. All three metal ions support this activity with Co(2+)> Mn(2+) congruent with Mg(2+). 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 in 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 analysis the rate of pyrophosphorolytic dismutase activity was found to be only moderately slower than the poly-merization activity. These results may help 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 pre-T cells.
Anderson
R S
RS
Baylor College of Medicine, Department of Biochemistry, One Baylor Plaza, Houston TX 77030, USA. randerson@masters.edu
Bollum
F J
FJ
Beattie
K L
KL
D000818:Animals; D002021:Buffers; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000235:genetics / Q000378:metabolism; D054306:Dideoxynucleotides; D011756:Diphosphates / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism*; D011108:Polymers; D011755:Pyrophosphatases / Q000378:metabolism; D013379:Substrate Specificity
D002021:Buffers; D002413:Cations, Divalent; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D011756:Diphosphates; D008670:Metals; D009838:Oligodeoxyribonucleotides; D011108:Polymers; C037773:2',3'-dideoxyadenosine triphosphate; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase