BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Recombination Activating Gene 1 Product Alone Possesses Endonucleolytic Activity

  • Kim, Deok-Ryong (Department of Biochemistry, College of Medicine, Gyeongsang National University)
  • Published : 2003.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Two lymphoid-specific proteins, RAG1 and RAG2, are required for the initiation of the V(D)J recombination in vitro. The V(D)J cleavage that is mediated by RAG proteins at the border between the coding and signal sequences results in the production of a hairpin at the coding end and a double-stranded break at the signal end. Two hairpin coding ends are re-opened, modified, and sealed; whereas, the signal ends are directly ligated. Here I report that only RAG1 can carry out a distinct endonucleolytic activity in vitro using an oligonucleotide substrate that is tethered by a short single-stranded DNA. The purified RAG1 protein alone formed a nick at the near position to the recombination signal sequence. This endonucleolytic activity was eliminated by immunoprecipitation using the RAG1-specific antibody, and required the 3'-hydroxy group. All of the RAG1 mutants that were incapable of the nick and hairpin formation in the V(D)J cleavage analysis also showed this new endonucleolytic activity. This suggests that the nicking activity that was observed might be functionally different from the nick formation in the V(D)J cleavage.

Keywords

References

  1. Akamatsu, Y. and Oettinger, M. A. (1998) Distinct roles of RAG1 and RAG2 in binding the V(D)J recombination signal sequences. Mol. Cell. Biol. 18, 4670-4678.
  2. Bailin, T., Mo, X. and Sadofsky, M. (1999) RAG1 and RAG2 tetramer complex is active in cleavage in V(D)J recombination. Mol. Cell. Biol. 19, 4664-4671.
  3. Bae, J. and Lee, S. (2001) The Human PTK6 Interacts with a 23- kDa Tyrosine-Phosphorylated Protein and is localized in Cytoplasm in Breast Carcinoma T-47D Cells. J. Biochem. Mol. Biol. 34, 33-38.
  4. Besmer, E., Mansilla-Soto, J., Cassard, S., Sawchuk, D. J., Brown. G., Sadofsky, M., Lewis, S. M., Nussenzweig, M. C. and Cortes, P. (1998) Hairpin coding end opening is mediated by RAG1 and RAG2 proteins. Mol. Cell 2, 817-828. https://doi.org/10.1016/S1097-2765(00)80296-8
  5. Cuomo, C. A., Mundy, C. L. and Oettinger. M. A. (1996) DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences. Mol. Cell. Biol. 16, 5683-5690.
  6. Eastman, Q. M., Leu, T. M. and Schatz, D. G. (1996) Initiation of Y(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85-88. https://doi.org/10.1038/380085a0
  7. Fugmann, S. and Schatz, D. (2001) Identification of basic residues in RAG2 critical for DNA binding by the RAG1-RAG2 complex. Mol. Cell 8, 889-910.
  8. Grawunder, U., Zimmer, D., Fugmann, S., Schwarz, K. and Lieber, M. R. (1998) DNA ligase IV is essential for Y(D)J recombination and DNA double-strand break repair in human precursor lymphocytes. Mol. Cell 2, 477-484. https://doi.org/10.1016/S1097-2765(00)80147-1
  9. Hammarsten, O. and Chu, G. (1998) DNA-dependent protein kinase: DNA binding and activation in the absence of Ku. Proc. Natl. Acad. Sci. USA 95, 525-530. https://doi.org/10.1073/pnas.95.2.525
  10. Hiom, K. and Gellert, M. (l997) A stable RAG1-RAG2-DNA complex that is active in Y(O)J cleavage. Cell 88, 65-72. https://doi.org/10.1016/S0092-8674(00)81859-0
  11. Kennedy, A., Guhathakurta, A., Kleckner, N., and Haniford, D. (1998) Tn 10 transposition via a DNA hairpin intermediate. Cell 95, 125-134. https://doi.org/10.1016/S0092-8674(00)81788-2
  12. Kim, D. R. (2001) Detennination of monoclonal antibodies capable of recognizing the native protein using surface plasma resonance. J. Biochem. Mol. Biol. 34, 452-456.
  13. Kim, D. R., Dai, Y., Mundy, C. L., Yang, W. and Oettinger, M. A. (1999) Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase. Genes Dev. 13, 3070-3080. https://doi.org/10.1101/gad.13.23.3070
  14. Kim, D. R. and Oettinger, M. A. (1998) Functional analysis of coordinated cleavage in V(D)J recombination. Mol. Cell BioI. 18, 4679-4688.
  15. Kim, D. R., Park, S. J. and Oettinger, M. A (2000) V(D)J recombination: site-specific cleavage and repair. Mol. Cells 10, 367-374.
  16. Landree, M. A., Wibbenmeyer, J. A. and Roth, D. B. (1999) Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination. Genes Dev. 13, 3059-3069. https://doi.org/10.1101/gad.13.23.3059
  17. Lee, S. (2001) Recognition of DNA damage in mammals. J. Biochem. Mol. Biol. 34, 489-495.
  18. Lewis, S. M. (1994) The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analysis. Adv. Immunol. 56, 27-50. https://doi.org/10.1016/S0065-2776(08)60450-2
  19. Li, Z., Otevrel, T., Gao, Y., Cheng, H., Seed, B., Stamato, T. D., Taccioli, G. E. and Alt, F. W. (1995) The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 83, 1079-1089. https://doi.org/10.1016/0092-8674(95)90135-3
  20. Ma, Y., Pannicke, U., Schwarz, K. and Lieber, M. R. (2002) Hairpin opening and overhang processing by an ArtemislDNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108, 781-794. https://doi.org/10.1016/S0092-8674(02)00671-2
  21. McBlane, J. E, van Gent, D. C., Ramsden, D. A, Romeo, C., Cuomo, C. A. and Gellert, M. (1995) Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83, 387-395. https://doi.org/10.1016/0092-8674(95)90116-7
  22. McElhinny, S. A., Snowden, C. M., McCarville, J. and Ramsden, D. A. (2000) Ku recruits the XRCC4-LigaseIV complex to DNA ends. Mol. Cell. Biol. 20, 2996-3003. https://doi.org/10.1128/MCB.20.9.2996-3003.2000
  23. Mo, X., Bailin, T. and Sadofsky, M. J. (1999) RAG1 and RAG2 cooperate in specific binding to the recombination signal sequence in vitro. J. Biol. Chem. 274, 7025-7031. https://doi.org/10.1074/jbc.274.11.7025
  24. Mombaerts, P., Iacomini, J., Johnson, R. S., Herrup, K, Furley, A. J. W. and Papaioannou, V. E. (1992) RAG-1 deficient mice have no mature B and T lymphocytes. Cell 68, 869-877. https://doi.org/10.1016/0092-8674(92)90030-G
  25. Oettinger, M. A., Schatz, D. G., Gorka, C. and Baltimore, D. (1990) RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248, 1517-1523. https://doi.org/10.1126/science.2360047
  26. Paull and Gellert (1999) Nbs1 potentiates ATP-driven DNA unwinding and endonuclesase cleavage by the Mre11/Rad 50 complex. Genes Dev. 13, 1276-1288. https://doi.org/10.1101/gad.13.10.1276
  27. Ramsden, D. A., McBlane, J. F., van Gent, D. C. and Gellert, M. (1996) Distinct DNA sequence and structure requirements for the two steps of V(D)J recombination signal cleavage. EMBO J.15, 3197-3206.
  28. Ramsden, D. A, Paull, T. T. and Gellert, M. (1997) Cell-free V(D)J recombination. Nature 388, 488-491. https://doi.org/10.1038/41351
  29. Sakano, H., Huppi, K, Heinrich, G. and Tonegawa, S. (1979) Sequences at the somatic recombination sites of immunoglobulin light chain genes. Nature 280, 288-294. https://doi.org/10.1038/280288a0
  30. Schatz, D. G., Oettinger, M. A. and Baltimore, D. (1989) The V(D)J recombination activating gene, RAG-1. Cell 59, 1035-1048. https://doi.org/10.1016/0092-8674(89)90760-5
  31. Shockett and Schatz (1999) DNA hairpin opening mediated by the RAG1 and RAG2 proteins. Mol. Cell. Biol. 19, 4159-4166
  32. Tonegawa, S. (1983) Somatic generation of antibody diversity. Nature 302, 575-581. https://doi.org/10.1038/302575a0
  33. Van Gent, D. C., McBlane, J. F., Ramsden, D. A, Sadofsky, M. J., Hesse, J. E. and Gellert, M. (1995) Initiation of V(D)J recombination in a cell-free system. Cell 81, 925-934. https://doi.org/10.1016/0092-8674(95)90012-8
  34. Van Gent, D. C., Ramsden, D. A. and Gellert, M. (1996) The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination. Cell 85, 107-113. https://doi.org/10.1016/S0092-8674(00)81086-7

Cited by

  1. Contribution of Rag1 to spatial memory ability in rats vol.236, 2013, https://doi.org/10.1016/j.bbr.2012.09.002