Skip to main content

Advertisement

SpringerLink
Log in

Characterization of terminal deoxynucleotidyl transferase and polymerase μ in zebrafish

  • Original Paper
  • Published:
Immunogenetics Aims and scope Submit manuscript

Abstract

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase μ (polμ), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and polμ and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish polμ. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Polμ expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Polμ is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and polμ in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and polμ provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and polμ themselves.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aoufouchi S, Flatter E, Dahan A, Faili A, Bertocci B, Storck S, Delbos F, Cocea L, Gupta N, Weill JC, Reynaud CA (2000) Two novel human and mouse DNA polymerases of the polX family. Nucleic Acids Res 28:3684–3693

    Article  PubMed  CAS  Google Scholar 

  • Bartl S, Miracle AL, Rumfelt LL, Kepler TB, Mochon E, Litman GW, Flajnik MF (2003) Terminal deoxynucleotidyl transferases from elasmobranchs reveal structural conservation within vertebrates. Immunogenetics 55:594–604

    Article  PubMed  CAS  Google Scholar 

  • Bassing CH, Swat W, Alt FW (2002) The mechanism and regulation of chromosomal V(D)J recombination. Cell 109 Suppl:S45–S55

    Article  PubMed  Google Scholar 

  • Bertocci B, De Smet A, Flatter E, Dahan A, Bories JC, Landreau C, Weill JC, Reynaud CA (2002) Cutting edge: DNA polymerases mu and lambda are dispensable for Ig gene hypermutation. J Immunol 168:3702–3706

    PubMed  CAS  Google Scholar 

  • Bertocci B, De Smet A, Berek C, Weill JC, Reynaud CA (2003) Immunoglobulin kappa light chain gene rearrangement is impaired in mice deficient for DNA polymerase mu. Immunity 19:203–211

    Article  PubMed  CAS  Google Scholar 

  • Bertocci B, De Smet A, Weill JC, Reynaud CA (2006) Nonoverlapping functions of DNA polymerases mu, lambda, and terminal deoxynucleotidyltransferase during immunoglobulin V(D)J recombination in vivo. Immunity 25:31–41

    Article  PubMed  CAS  Google Scholar 

  • Bollum FJ (1974) Terminal deoxynucleotidyl transferase. Enzymes 10:145–171

    CAS  Google Scholar 

  • Bork P, Hofmann K, Bucher P, Neuwald AF, Altschul SF, Koonin EV (1997) A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J 11:68–76

    PubMed  CAS  Google Scholar 

  • Breitschopf H, Suchanek G, Gould RM, Colman DR, Lassmann H (1992) In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol (Berl) 84:581–587

    Article  CAS  Google Scholar 

  • Burma S, Chen BP, Chen DJ (2006) Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst) 5:1042–1048

    Article  CAS  Google Scholar 

  • Danilova N, Bussmann J, Jekosch K, Steiner LA (2005) The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z. Nat Immunol 6:295–302

    Article  PubMed  CAS  Google Scholar 

  • Danilova N, Hohman VS, Kim EH, Steiner LA (2000) Immunoglobulin variable-region diversity in the zebrafish. Immunogenetics 52:81–91

    Article  PubMed  CAS  Google Scholar 

  • Danilova N, Hohman VS, Sacher F, Ota T, Willett CE, Steiner LA (2004) T cells and the thymus in developing zebrafish. Dev Comp Immunol 28:755–767

    Article  PubMed  CAS  Google Scholar 

  • Danilova N, Steiner LA (2002) B cells develop in the zebrafish pancreas. Proc Natl Acad Sci U S A 99:13711–13716

    Article  PubMed  CAS  Google Scholar 

  • Davies JF 2nd, Almassy RJ, Hostomska Z, Ferre RA, Hostomsky Z (1994) 2.3 A crystal structure of the catalytic domain of DNA polymerase beta. Cell 76:1123–1133

    Article  PubMed  CAS  Google Scholar 

  • Delarue M, Boule JB, Lescar J, Expert-Bezancon N, Jourdan N, Sukumar N, Rougeon F, Papanicolaou C (2002) Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase. EMBO J 21:427–439

    Article  PubMed  CAS  Google Scholar 

  • Desiderio SV, Yancopoulos GD, Paskind M, Thomas E, Boss MA, Landau N, Alt FW, Baltimore D (1984) Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells. Nature 311:752–755

    Article  PubMed  CAS  Google Scholar 

  • Doherty AJ, Serpell LC, Ponting CP (1996) The helix-hairpin-helix DNA-binding motif: a structural basis for non-sequence-specific recognition of DNA. Nucleic Acids Res 24:2488–2497

    Article  PubMed  CAS  Google Scholar 

  • Dominguez O, Ruiz JF, Lain de Lera T, Garcia-Diaz M, Gonzalez MA, Kirchhoff T, Martinez AC, Bernad A, Blanco L (2000) DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells. EMBO J 19:1731–1742

    Article  PubMed  CAS  Google Scholar 

  • Doublie S, Sawaya MR, Ellenberger T (1999) An open and closed case for all polymerases. Structure Fold Des 7:R31–35

    Article  PubMed  CAS  Google Scholar 

  • Doyen N, d’Andon MF, Bentolila LA, Nguyen QT, Rougeon F (1993) Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase. Nucleic Acids Res 21:1187–1191

    Article  PubMed  CAS  Google Scholar 

  • Gilfillan S, Benoist C, Mathis D (1995) Mice lacking terminal deoxynucleotidyl transferase: adult mice with a fetal antigen receptor repertoire. Immunol Rev 148:201–219

    Article  PubMed  CAS  Google Scholar 

  • Gilfillan S, Dierich A, Lemeur M, Benoist C, Mathis D (1993) Mice lacking TdT: mature animals with an immature lymphocyte repertoire. Science 261:1175–1178

    Article  PubMed  CAS  Google Scholar 

  • Glover JN, Williams RS, Lee MS (2004) Interactions between BRCT repeats and phosphoproteins: tangled up in two. Trends Biochem Sci 29:579–585

    Article  PubMed  CAS  Google Scholar 

  • Golub R, Andre S, Hassanin A, Affaticati P, Larijani M, Fellah JS (2004) Early expression of two TdT isoforms in the hematopoietic system of the Mexican axolotl: implications for the evolutionary origin of the N-nucleotide addition. Immunogenetics 56:204–213

    Article  PubMed  CAS  Google Scholar 

  • Guth AM, Rosenberg GH, Miller RD (1998) Opossum (Monodelphis domestica) terminal deoxynucleotidyl transferase gene. Immunogenetics 47:483–486

    Article  PubMed  CAS  Google Scholar 

  • Hansen JD (1997) Characterization of rainbow trout terminal deoxynucleotidyl transferase structure and expression. TdT and RAG1 co-expression define the trout primary lymphoid tissues. Immunogenetics 46:367–375

    Article  PubMed  CAS  Google Scholar 

  • Holm L, Sander C (1995) DNA polymerase beta belongs to an ancient nucleotidyltransferase superfamily. Trends Biochem Sci 20:345–347

    Article  PubMed  CAS  Google Scholar 

  • Juarez R, Ruiz JF, McElhinny SA, Ramsden D, Blanco L (2006) A specific loop in human DNA polymerase mu allows switching between creative and DNA-instructed synthesis. Nucleic Acids Res 34:4572–4582

    Article  PubMed  CAS  Google Scholar 

  • Koiwai O, Yokota T, Kageyama T, Hirose T, Yoshida S, Arai K (1986) Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells. Nucleic Acids Res 14:5777–5792

    Article  PubMed  CAS  Google Scholar 

  • Komori T, Okada A, Stewart V, Alt FW (1993) Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. Science 261:1171–1175

    Article  PubMed  CAS  Google Scholar 

  • Lam SH, Chua HL, Gong Z, Lam TJ, Sin YM (2004) Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study. Dev Comp Immunol 28:9–28

    Article  PubMed  CAS  Google Scholar 

  • Lee A, Hsu E (1994) Isolation and characterization of the Xenopus terminal deoxynucleotidyl transferase. J Immunol 152:4500–4507

    PubMed  CAS  Google Scholar 

  • Lucas D, Lain de Lera T, Gonzalez MA, Ruiz JF, Dominguez O, Casanova JC, Martinez AC, Blanco L, Bernad A (2005) Polymerase mu is up-regulated during the T cell-dependent immune response and its deficiency alters developmental dynamics of spleen centroblasts. Eur J Immunol 35:1601–1611

    Article  PubMed  CAS  Google Scholar 

  • Ma Y, Lu H, Tippin B, Goodman MF, Shimazaki N, Koiwai O, Hsieh CL, Schwarz K, Lieber MR (2004) A biochemically defined system for mammalian nonhomologous DNA end joining. Mol Cell 16:701–713

    Article  PubMed  CAS  Google Scholar 

  • Maga G, Hubscher U (2003) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci 116:3051–3060

    Article  PubMed  CAS  Google Scholar 

  • Mahajan KN, Gangi-Peterson L, Sorscher DH, Wang J, Gathy KN, Mahajan NP, Reeves WH, Mitchell BS (1999) Association of terminal deoxynucleotidyl transferase with Ku. Proc Natl Acad Sci U S A 96:13926–13931

    Article  PubMed  CAS  Google Scholar 

  • Mahajan KN, Nick McElhinny SA, Mitchell BS, Ramsden DA (2002) Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair. Mol Cell Biol 22:5194–5202

    Article  PubMed  CAS  Google Scholar 

  • Miracle AL, Anderson MK, Litman RT, Walsh CJ, Luer CA, Rothenberg EV, Litman GW (2001) Complex expression patterns of lymphocyte-specific genes during the development of cartilaginous fish implicate unique lymphoid tissues in generating an immune repertoire. Int Immunol 13:567–580

    Article  PubMed  CAS  Google Scholar 

  • Nick McElhinny SA, Ramsden DA (2004) Sibling rivalry: competition between Pol×family members in V(D)J recombination and general double strand break repair. Immunol Rev 200:156–164

    Article  PubMed  Google Scholar 

  • Nick McElhinny SA, Havener JM, Garcia-Diaz M, Juarez R, Bebenek K, Kee BL, Blanco L, Kunkel TA, Ramsden DA (2005) A gradient of template dependence defines distinct biological roles for family×polymerases in nonhomologous end joining. Mol Cell 19:357–366

    Article  PubMed  CAS  Google Scholar 

  • Patel HM, Hsu E (1997) Abbreviated junctional sequences impoverish antibody diversity in urodele amphibians. J Immunol 159:3391–3399

    PubMed  CAS  Google Scholar 

  • Pelletier H, Sawaya MR (1996) Characterization of the metal ion binding helix-hairpin-helix motifs in human DNA polymerase beta by X-ray structural analysis. Biochemistry 35:12778–12787

    Article  PubMed  CAS  Google Scholar 

  • Pelletier H, Sawaya MR, Kumar A, Wilson SH, Kraut J (1994) Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science 264:1891–1903

    Article  PubMed  CAS  Google Scholar 

  • Pelletier H, Sawaya MR, Wolfle W, Wilson SH, Kraut J (1996) Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity. Biochemistry 35:12742–12761

    Article  PubMed  CAS  Google Scholar 

  • Ramadan K, Shevelev I, Hubscher U (2004) The DNA-polymerase-X family: controllers of DNA quality. Nat Rev Mol Cell Biol 5:1038–1043

    Article  PubMed  CAS  Google Scholar 

  • Repasky JA, Corbett E, Boboila C, Schatz DG (2004) Mutational analysis of terminal deoxynucleotidyltransferase-mediated N-nucleotide addition in V(D)J recombination. J Immunol 172:5478–5488

    PubMed  CAS  Google Scholar 

  • Ruiz JF, Juarez R, Garcia-Diaz M, Terrados G, Picher AJ, Gonzalez-Barrera S, Fernandez de Henestrosa AR, Blanco L (2003) Lack of sugar discrimination by human Pol mu requires a single glycine residue. Nucleic Acids Res 31:4441–4449

    Article  PubMed  CAS  Google Scholar 

  • Ruiz JF, Lucas D, Garcia-Palomero E, Saez AI, Gonzalez MA, Piris MA, Bernad A, Blanco L (2004) Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt’s lymphoma cell line affects the somatic hypermutation rate. Nucleic Acids Res 32:5861–5873

    Article  PubMed  CAS  Google Scholar 

  • Rumfelt LL, Avila D, Diaz M, Bartl S, McKinney EC, Flajnik MF (2001) A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG. Proc Natl Acad Sci U S A 98:1775–1780

    Article  PubMed  CAS  Google Scholar 

  • Sawaya MR, Pelletier H, Kumar A, Wilson SH, Kraut J (1994) Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. Science 264:1930–1935

    Article  PubMed  CAS  Google Scholar 

  • Sawaya MR, Prasad R, Wilson SH, Kraut J, Pelletier H (1997) Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry 36:11205–11215

    Article  PubMed  CAS  Google Scholar 

  • Schorpp M, Bialecki M, Diekhoff D, Walderich B, Odenthal J, Maischein HM, Zapata AG, Boehm T (2006) Conserved functions of Ikaros in vertebrate lymphocyte development: genetic evidence for distinct larval and adult phases of T cell development and two lineages of B cells in zebrafish. J Immunol 177:2463–2476

    PubMed  CAS  Google Scholar 

  • Scott SP, Pandita TK (2007) The cellular control of DNA double-strand breaks. J Cell Biochem (in press)

  • Takahara K, Hayashi N, Fujita-Sagawa K, Morishita T, Hashimoto Y, Noda A (1994) Alternative splicing of bovine terminal deoxynucleotidyl transferase cDNA. Biosci Biotechnol Biochem 58:786–787

    Article  PubMed  CAS  Google Scholar 

  • Thai TH, Kearney JF (2005) Isoforms of terminal deoxynucleotidyltransferase: developmental aspects and function. Adv Immunol 86:113–136

    PubMed  CAS  Google Scholar 

  • Traver D, Herbomel P, Patton EE, Murphey RD, Yoder JA, Litman GW, Catic A, Amemiya CT, Zon LI, Trede NS (2003) The zebrafish as a model organism to study development of the immune system. Adv Immunol 81:253–330

    PubMed  Google Scholar 

  • Westerfield M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene

    Google Scholar 

  • Willett CE, Cherry JJ, Steiner LA (1997a) Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish. Immunogenetics 45:394–404

    Article  CAS  Google Scholar 

  • Willett CE, Zapata AG, Hopkins N, Steiner LA (1997b) Expression of zebrafish rag genes during early development identifies the thymus. Dev Biol 182:331–341

    Article  CAS  Google Scholar 

  • Willett CE, Kawasaki H, Amemiya CT, Lin S, Steiner LA (2001) Ikaros expression as a marker for lymphoid progenitors during zebrafish development. Dev Dyn 222:694–698

    Article  PubMed  CAS  Google Scholar 

  • Yang B, Gathy KN, Coleman MS (1994) Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase. J Biol Chem 269:11859–11868

    PubMed  CAS  Google Scholar 

  • Yang B, Gathy KN, Coleman MS (1995) T-cell specific avian TdT: characterization of the cDNA and recombinant enzyme. Nucleic Acids Res 23:2041–2048

    Article  PubMed  CAS  Google Scholar 

  • Zapata A, Amemiya CT (2000) Phylogeny of lower vertebrates and their immunological structures. Curr Top Microbiol Immunol 248:67–107

    PubMed  CAS  Google Scholar 

Download references

Acknowledgment

This work was supported by the National Institutes of Health (R01 AI08054). Many thanks to Michael Schorpp and Leonard Lerman for fruitful discussions.

Author information

Authors and Affiliations

  1. Biology Department, Massachusetts Institute of Technology, Cambridge, MA, USA

    Susann Beetz & Lisa A. Steiner

  2. Department of Developmental Immunology, Max-Planck-Institute of Immunobiology, Freiburg, Germany

    Dagmar Diekhoff

  3. Institute of Immunology, University Hospital Schleswig-Holstein Campus Kiel, Michaelisstr. 5, 24105, Kiel, Germany

    Susann Beetz

Authors
  1. Susann Beetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dagmar Diekhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lisa A. Steiner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Susann Beetz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Beetz, S., Diekhoff, D. & Steiner, L.A. Characterization of terminal deoxynucleotidyl transferase and polymerase μ in zebrafish. Immunogenetics 59, 735–744 (2007). https://doi.org/10.1007/s00251-007-0241-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00251-007-0241-7

Keywords

Search

Navigation