Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Aug;17(8):4544–4552. doi: 10.1128/mcb.17.8.4544

A basic motif in the N-terminal region of RAG1 enhances V(D)J recombination activity.

C J McMahan 1, M J Difilippantonio 1, N Rao 1, E Spanopoulou 1, D G Schatz 1
PMCID: PMC232308  PMID: 9234712

Abstract

The variable portions of antigen receptor genes are assembled from component gene segments by a site-specific recombination reaction known as V(D)J recombination. The RAG1 and RAG2 proteins are the critical lymphoid cell-specific components of the recombination enzymatic machinery and are responsible for site-specific DNA recognition and cleavage. Previous studies had defined a minimal, recombinationally active core region of murine RAG1 consisting of amino acids 384 to 1008 of the 1,040-residue RAG1 protein. No recombination function has heretofore been ascribed to any portion of the 383-amino-acid N-terminal region that is missing from the core, but it seems likely to be of functional significance, based on its evolutionary conservation. Using extrachromosomal recombination substrates, we demonstrate here that the N-terminal region enhances the recombination activity of RAG1 by up to an order of magnitude in a variety of cell lines. Deletion analysis localized a region of the N terminus critical for this effect to amino acids 216 to 238, and further mutagenesis demonstrated that a small basic amino acid motif (BIIa) in this region is essential for enhancing the activity of RAG1. Despite the fact that BIIa is important for the interaction of RAG1 with the nuclear localization factor Srp-1, it does not appear to enhance recombination by facilitating nuclear transport of RAG1. A variety of models for how this region stimulates the recombination activity of RAG1 are considered.

Full Text

The Full Text of this article is available as a PDF (667.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agrawal A., Schatz D. G. RAG1 and RAG2 form a stable postcleavage synaptic complex with DNA containing signal ends in V(D)J recombination. Cell. 1997 Apr 4;89(1):43–53. doi: 10.1016/s0092-8674(00)80181-6. [DOI] [PubMed] [Google Scholar]
  2. Bogue M., Roth D. B. Mechanism of V(D)J recombination. Curr Opin Immunol. 1996 Apr;8(2):175–180. doi: 10.1016/s0952-7915(96)80055-0. [DOI] [PubMed] [Google Scholar]
  3. Cuomo C. A., Mundy C. L., Oettinger M. A. DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences. Mol Cell Biol. 1996 Oct;16(10):5683–5690. doi: 10.1128/mcb.16.10.5683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Difilippantonio M. J., McMahan C. J., Eastman Q. M., Spanopoulou E., Schatz D. G. RAG1 mediates signal sequence recognition and recruitment of RAG2 in V(D)J recombination. Cell. 1996 Oct 18;87(2):253–262. doi: 10.1016/s0092-8674(00)81343-4. [DOI] [PubMed] [Google Scholar]
  5. Eastman Q. M., Leu T. M., Schatz D. G. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature. 1996 Mar 7;380(6569):85–88. doi: 10.1038/380085a0. [DOI] [PubMed] [Google Scholar]
  6. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Freemont P. S., Hanson I. M., Trowsdale J. A novel cysteine-rich sequence motif. Cell. 1991 Feb 8;64(3):483–484. doi: 10.1016/0092-8674(91)90229-r. [DOI] [PubMed] [Google Scholar]
  8. Gauss G. H., Lieber M. R. Unequal signal and coding joint formation in human V(D)J recombination. Mol Cell Biol. 1993 Jul;13(7):3900–3906. doi: 10.1128/mcb.13.7.3900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Görlich D., Prehn S., Laskey R. A., Hartmann E. Isolation of a protein that is essential for the first step of nuclear protein import. Cell. 1994 Dec 2;79(5):767–778. doi: 10.1016/0092-8674(94)90067-1. [DOI] [PubMed] [Google Scholar]
  10. Hesse J. E., Lieber M. R., Gellert M., Mizuuchi K. Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals. Cell. 1987 Jun 19;49(6):775–783. doi: 10.1016/0092-8674(87)90615-5. [DOI] [PubMed] [Google Scholar]
  11. Hiom K., Gellert M. A stable RAG1-RAG2-DNA complex that is active in V(D)J cleavage. Cell. 1997 Jan 10;88(1):65–72. doi: 10.1016/s0092-8674(00)81859-0. [DOI] [PubMed] [Google Scholar]
  12. Kirch S. A., Sudarsanam P., Oettinger M. A. Regions of RAG1 protein critical for V(D)J recombination. Eur J Immunol. 1996 Apr;26(4):886–891. doi: 10.1002/eji.1830260425. [DOI] [PubMed] [Google Scholar]
  13. LaCasse E. C., Lefebvre Y. A. Nuclear localization signals overlap DNA- or RNA-binding domains in nucleic acid-binding proteins. Nucleic Acids Res. 1995 May 25;23(10):1647–1656. doi: 10.1093/nar/23.10.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leu T. M., Schatz D. G. rag-1 and rag-2 are components of a high-molecular-weight complex, and association of rag-2 with this complex is rag-1 dependent. Mol Cell Biol. 1995 Oct;15(10):5657–5670. doi: 10.1128/mcb.15.10.5657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lewis S. M., Hesse J. E. Cutting and closing without recombination in V(D)J joining. EMBO J. 1991 Dec;10(12):3631–3639. doi: 10.1002/j.1460-2075.1991.tb04929.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lewis S. M. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv Immunol. 1994;56:27–150. doi: 10.1016/s0065-2776(08)60450-2. [DOI] [PubMed] [Google Scholar]
  17. Lin W. C., Desiderio S. Regulation of V(D)J recombination activator protein RAG-2 by phosphorylation. Science. 1993 May 14;260(5110):953–959. doi: 10.1126/science.8493533. [DOI] [PubMed] [Google Scholar]
  18. Livak F., Schatz D. G. T-cell receptor alpha locus V(D)J recombination by-products are abundant in thymocytes and mature T cells. Mol Cell Biol. 1996 Feb;16(2):609–618. doi: 10.1128/mcb.16.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McBlane J. F., van Gent D. C., Ramsden D. A., Romeo C., Cuomo C. A., Gellert M., Oettinger M. A. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell. 1995 Nov 3;83(3):387–395. doi: 10.1016/0092-8674(95)90116-7. [DOI] [PubMed] [Google Scholar]
  20. McMahan C. J., Sadofsky M. J., Schatz D. G. Definition of a large region of RAG1 that is important for coimmunoprecipitation of RAG2. J Immunol. 1997 Mar 1;158(5):2202–2210. [PubMed] [Google Scholar]
  21. Melchior F., Gerace L. Mechanisms of nuclear protein import. Curr Opin Cell Biol. 1995 Jun;7(3):310–318. doi: 10.1016/0955-0674(95)80084-0. [DOI] [PubMed] [Google Scholar]
  22. Ramsden D. A., Gellert M. Formation and resolution of double-strand break intermediates in V(D)J rearrangement. Genes Dev. 1995 Oct 1;9(19):2409–2420. doi: 10.1101/gad.9.19.2409. [DOI] [PubMed] [Google Scholar]
  23. Ramsden D. A., McBlane J. F., van Gent D. C., Gellert M. Distinct DNA sequence and structure requirements for the two steps of V(D)J recombination signal cleavage. EMBO J. 1996 Jun 17;15(12):3197–3206. [PMC free article] [PubMed] [Google Scholar]
  24. Rodgers K. K., Bu Z., Fleming K. G., Schatz D. G., Engelman D. M., Coleman J. E. A zinc-binding domain involved in the dimerization of RAG1. J Mol Biol. 1996 Jul 5;260(1):70–84. doi: 10.1006/jmbi.1996.0382. [DOI] [PubMed] [Google Scholar]
  25. Sadofsky M. J., Hesse J. E., McBlane J. F., Gellert M. Expression and V(D)J recombination activity of mutated RAG-1 proteins. Nucleic Acids Res. 1993 Dec 11;21(24):5644–5650. doi: 10.1093/nar/21.24.5644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Saurin A. J., Borden K. L., Boddy M. N., Freemont P. S. Does this have a familiar RING? Trends Biochem Sci. 1996 Jun;21(6):208–214. [PubMed] [Google Scholar]
  27. Schatz D. G., Baltimore D. Stable expression of immunoglobulin gene V(D)J recombinase activity by gene transfer into 3T3 fibroblasts. Cell. 1988 Apr 8;53(1):107–115. doi: 10.1016/0092-8674(88)90492-8. [DOI] [PubMed] [Google Scholar]
  28. Schatz D. G., Oettinger M. A., Baltimore D. The V(D)J recombination activating gene, RAG-1. Cell. 1989 Dec 22;59(6):1035–1048. doi: 10.1016/0092-8674(89)90760-5. [DOI] [PubMed] [Google Scholar]
  29. Silver D. P., Spanopoulou E., Mulligan R. C., Baltimore D. Dispensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V(D)J recombination. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6100–6104. doi: 10.1073/pnas.90.13.6100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Spanopoulou E., Cortes P., Shih C., Huang C. M., Silver D. P., Svec P., Baltimore D. Localization, interaction, and RNA binding properties of the V(D)J recombination-activating proteins RAG1 and RAG2. Immunity. 1995 Dec;3(6):715–726. doi: 10.1016/1074-7613(95)90061-6. [DOI] [PubMed] [Google Scholar]
  31. Spanopoulou E., Zaitseva F., Wang F. H., Santagata S., Baltimore D., Panayotou G. The homeodomain region of Rag-1 reveals the parallel mechanisms of bacterial and V(D)J recombination. Cell. 1996 Oct 18;87(2):263–276. doi: 10.1016/s0092-8674(00)81344-6. [DOI] [PubMed] [Google Scholar]
  32. Weaver D. T. What to do at an end: DNA double-strand-break repair. Trends Genet. 1995 Oct;11(10):388–392. doi: 10.1016/s0168-9525(00)89121-0. [DOI] [PubMed] [Google Scholar]
  33. Weis K., Mattaj I. W., Lamond A. I. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science. 1995 May 19;268(5213):1049–1053. doi: 10.1126/science.7754385. [DOI] [PubMed] [Google Scholar]
  34. van Gent D. C., McBlane J. F., Ramsden D. A., Sadofsky M. J., Hesse J. E., Gellert M. Initiation of V(D)J recombination in a cell-free system. Cell. 1995 Jun 16;81(6):925–934. doi: 10.1016/0092-8674(95)90012-8. [DOI] [PubMed] [Google Scholar]
  35. van Gent D. C., Ramsden D. A., Gellert M. The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination. Cell. 1996 Apr 5;85(1):107–113. doi: 10.1016/s0092-8674(00)81086-7. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES