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Distinct and opposite diversifying activities of terminal transferase splice variants

Nature Immunology volume 3, pages 457462 (2002) | Download Citation

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Abstract

The short splice variant of mouse terminal deoxynucleotidyl transferase (TdTS) catalyzes the addition of nontemplated nucleotides (N addition) at the coding joins of B cell and T cell antigen receptor genes. However, the activity and function of the long isoform of TdT (TdTL) have not been determined. We show here, in vitro and in vivo, that TdTL is a 3′→5′ exonuclease that catalyzes the deletion of nucleotides at coding joins. These findings suggest that the two TdT isoforms may act in concert to preserve the integrity of the variable region of antigen receptors while generating diversity.

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References

  1. 1.

    & Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc. Natl. Acad. Sci. USA 73, 3628–3632 (1976).

  2. 2.

    The mechanism of V(D)J joining: Lessons from molecular, immunological, and comparative analyses. Adv. Immunol. 56, 27–150 (1994).

  3. 3.

    et al. The RAG proteins and V(D)J recombination: complexes, ends, and transposition. Ann. Rev. Immunol. 18, 497–527 (2000).

  4. 4.

    V(D)J recombination: on the cutting edge. Curr. Opin. Cell Biol. 11, 325–329 (1999).

  5. 5.

    et al. A direct estimate of the human αβ T cell receptor diversity. Science 286, 958–961 (1999).

  6. 6.

    et al. Most αβ T cell receptor diversity is due to terminal deoxynucleotidyl transferase. J. Exp. Med. 194, 1385–1390 (2001).

  7. 7.

    , , & Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. Science 261, 1171–1175 (1993).

  8. 8.

    , , , & Mice lacking TdT: mature animals with an immature lymphocyte repertoire. Science 261, 1175–1178 (1993).

  9. 9.

    et al. Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase. Nucleic Acids Res. 21, 1187–1191 (1993).

  10. 10.

    et al. Isolation and characterization of bovine and mouse terminal deoxynucleotidyl transferase cDNAs expressible in mammalian cells. Nucleic Acids Res. 14, 5777–5792 (1986).

  11. 11.

    , , & Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: identification of peptides in the DNA binding domain. Biochemistry 30, 3075–3082 (1991).

  12. 12.

    , & Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase. J. Biol. Chem. 269, 11859–11868 (1994).

  13. 13.

    & Increased junctional diversity in fetal B cells results in a loss of protective anti-phosphorylcholine antibodies in adult mice. Immunity 5, 607–617 (1999).

  14. 14.

    et al. The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization. EMBO J. 14, 4221–4229 (1995).

  15. 15.

    Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences. J. Exp. Med. 172, 1377–1390 (1990).

  16. 16.

    , , , & Junctional sequences of T cell receptor γδ genes: implications for γδ T cell lineages and for a novel intermediate of V-(D)-J joining. Cell 59, 859–870 (1989).

  17. 17.

    , , & Regulated expression and structure of T cell receptor γ/δ transcripts in human thymic ontogeny. EMBO J. 10, 83–91 (1991).

  18. 18.

    , , & The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus. EMBO J. 10, 2461–2470 (1991).

  19. 19.

    , & Developmentally controlled selection of antibody genes: characterization of individual VH7183 genes and evidence for stage-specific somatic diversification. Eur. J. Immunol. 22, 71–78 (1992).

  20. 20.

    & Restricted κ chain expression in early ontogeny: biased utilization of Vκ exons and preferential Vκ-Jκ recombinations. J. Exp. Med. 177, 1317–1330 (1993).

  21. 21.

    & Clonal proliferation unlinked to terminal deoxynucleotidyl transferase synthesis in thymocytes of young mice. J. Immunol. 130, 1627–1633 (1983).

  22. 22.

    , & The long isoform of terminal deoxynucleotidyl transferase enters the nucleus and, rather than catalyzing nontemplated nucleotide addition, modulates the catalytic activity of the short isoform. J. Exp. Med. 193, 89–99 (2001).

  23. 23.

    et al. Positive and negative selection events during B lymphopoiesis. Curr. Opin. Immunol. 7, 214–227 (1995).

  24. 24.

    et al. Down-regulation of RAG1 and RAG2 gene expression in pre-B cells after functional immunoglobulin heavy chain rearrangement. Immunity 3, 601–608 (1995).

  25. 25.

    & Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3′→5′ exonucleases. J. Biol. Chem. 274, 19655–19660 (1999).

  26. 26.

    Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination. Mol. Cell Biol. 18, 2029–2037 (1998).

  27. 27.

    & Identification of V(D)J recombination coding end intermediates in normal thymocytes. J. Mol. Biol. 267, 1–9 (1997).

  28. 28.

    , & Characterization of broken DNA molecules associated with V(D)J recombination. Proc. Natl. Acad. Sci. USA 90, 10788–10972 (1993).

  29. 29.

    , , , & Double-strand signal sequence breaks in V(D)J recombination are blunt, 5′-phosphorylated, RAG-dependent, and cell-cycle regulated. Genes Dev. 7, 2520–2532 (1993).

  30. 30.

    et al. A human DNA editing enzyme homologous to the Escherichia coli Dna/Q/MutD protein. EMBO J. 18, 3868–3875 (1999).

  31. 31.

    , , , & A conserved 3′→5′ exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell 59, 219–228 (1989).

  32. 32.

    et al. DNA polymerization in the absence of exonucleolytic proofreading: In vivo and in vitro studies. Proc. Natl. Acad. Sci. USA 88, 2417–2421 (1991).

  33. 33.

    , & The 3′→5′ exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. EMBO J. 1, 17–24 (1991).

  34. 34.

    & Genetic and biochemical studies of bactriophage T4 DNA polymerase 3′→5′-exonuclease activity. J. Biol. Chem. 268, 27100–27108 (1983).

  35. 35.

    , , , & Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions. Biochemistry 35, 8110–8119 (1996).

  36. 36.

    , , & Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 29, 1638–1646 (2001).

  37. 37.

    et al. Alternative splicing of bovine terminal deoxynucleotidyl transferase cDNA. Biosci. Biotech. Biochem. 58, 786–787 (1994).

  38. 38.

    , & Comparison of the two murine deoxynucleotidyl transferase. J. Biol. Chem. 275, 28984–28988 (2000).

  39. 39.

    et al. Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-xL transgene. Immunity 4, 291–299 (1996).

  40. 40.

    , & Limited junctional diversity in κ light chains. J. Immnunol. 152, 3467 (1994).

  41. 41.

    & Rearrangement and selection in the developing Vκ repertoire of the mouse: an analysis of the usage of two Vκ gene. J. Immunol. 160, 4904–4913 (1998).

  42. 42.

    TdT effects during κ light chain recombination and influence on BCR selection. Thesis, Yale Univ. (2000).

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Acknowledgements

We thank S. Gilfillan and D. Mathis for the TdTL and TdTS cDNA clones; D. J. Mazure and F. W. Perrino for hTREX2; M. Oettinger for full-length RAG-1 and RAG-2; L. Gartland for FACS; X. Y. Liu for technical assistance; M. A. Anderson, D. S. Nelson, P. D. Burrows for helpful discussions; and A. Brookshire for preparing this manuscript. Supported by NIH grants AI 523133, AI 07051, AI36420 and the Howard Hughes Medical Institute (D. B. R.).

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Author notes

    • Mary M. Purugganan

    Present address: Cain Project in Engineering and Professional Communication, Rice University, P.O. Box 1892, MS345, Houston, TX 77251-1892, USA.

Affiliations

  1. Division of Developmental and Clinical Immunology, Department of Microbiology, The University of Alabama at Birmingham, 378 Wallace Tumor Institute, Birmingham, AL 35294, USA.

    • To-Ha Thai
    •  & John F. Kearney
  2. Department of Immunology, M929, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.

    • Mary M. Purugganan
    •  & David B. Roth

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The authors declare no competing financial interests.

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Correspondence to John F. Kearney.

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DOI

https://doi.org/10.1038/ni788