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. 1997 Nov;17(11):6379–6385. doi: 10.1128/mcb.17.11.6379

V(D)J recombination: in vitro coding joint formation.

F Weis-Garcia 1, E Besmer 1, D J Sawchuk 1, W Yu 1, Y Hu 1, S Cassard 1, M C Nussenzweig 1, P Cortes 1
PMCID: PMC232489  PMID: 9343399

Abstract

Antigen receptor genes are assembled through a mechanism known as V(D)J recombination, which involves two different joining reactions: signal and coding joining. Formation of these joints is essential for antigen receptor assembly as well as maintaining chromosomal integrity. Here we report on a cell-free system for coding joint formation using deletion and inversion recombination substrates. In vitro coding joint formation requires RAG1, RAG2, and heat-labile factors present in the nuclear extract of nonlymphoid cells. Both inversion- and deletion-mediated coding joint reactions produce diverse coding joints, with deletions and P nucleotide addition. We also show that deletion-mediated coding joint formation follows the 12/23 rule and requires the catalytic subunit of DNA-dependent protein kinase.

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Selected References

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  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. Blunt T., Finnie N. J., Taccioli G. E., Smith G. C., Demengeot J., Gottlieb T. M., Mizuta R., Varghese A. J., Alt F. W., Jeggo P. A. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell. 1995 Mar 10;80(5):813–823. doi: 10.1016/0092-8674(95)90360-7. [DOI] [PubMed] [Google Scholar]
  3. Cortes P., Weis-Garcia F., Misulovin Z., Nussenzweig A., Lai J. S., Li G., Nussenzweig M. C., Baltimore D. In vitro V(D)J recombination: signal joint formation. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14008–14013. doi: 10.1073/pnas.93.24.14008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Dignam J. D., Martin P. L., Shastry B. S., Roeder R. G. Eukaryotic gene transcription with purified components. Methods Enzymol. 1983;101:582–598. doi: 10.1016/0076-6879(83)01039-3. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Gauss G. H., Lieber M. R. Mechanistic constraints on diversity in human V(D)J recombination. Mol Cell Biol. 1996 Jan;16(1):258–269. doi: 10.1128/mcb.16.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Ge H., Martinez E., Chiang C. M., Roeder R. G. Activator-dependent transcription by mammalian RNA polymerase II: in vitro reconstitution with general transcription factors and cofactors. Methods Enzymol. 1996;274:57–71. doi: 10.1016/s0076-6879(96)74008-9. [DOI] [PubMed] [Google Scholar]
  11. Gilfillan S., Dierich A., Lemeur M., Benoist C., Mathis D. Mice lacking TdT: mature animals with an immature lymphocyte repertoire. Science. 1993 Aug 27;261(5125):1175–1178. doi: 10.1126/science.8356452. [DOI] [PubMed] [Google Scholar]
  12. Hendrickson E. A., Qin X. Q., Bump E. A., Schatz D. G., Oettinger M., Weaver D. T. A link between double-strand break-related repair and V(D)J recombination: the scid mutation. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4061–4065. doi: 10.1073/pnas.88.10.4061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hesse J. E., Lieber M. R., Mizuuchi K., Gellert M. V(D)J recombination: a functional definition of the joining signals. Genes Dev. 1989 Jul;3(7):1053–1061. doi: 10.1101/gad.3.7.1053. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Jackson S. P., Jeggo P. A. DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK. Trends Biochem Sci. 1995 Oct;20(10):412–415. doi: 10.1016/s0968-0004(00)89090-8. [DOI] [PubMed] [Google Scholar]
  17. Kallenbach S., Doyen N., Fanton d'Andon M., Rougeon F. Three lymphoid-specific factors account for all junctional diversity characteristic of somatic assembly of T-cell receptor and immunoglobulin genes. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2799–2803. doi: 10.1073/pnas.89.7.2799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kemp L. M., Sedgwick S. G., Jeggo P. A. X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining. Mutat Res. 1984 Nov-Dec;132(5-6):189–196. doi: 10.1016/0167-8817(84)90037-3. [DOI] [PubMed] [Google Scholar]
  19. Kirchgessner C. U., Patil C. K., Evans J. W., Cuomo C. A., Fried L. M., Carter T., Oettinger M. A., Brown J. M. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science. 1995 Feb 24;267(5201):1178–1183. doi: 10.1126/science.7855601. [DOI] [PubMed] [Google Scholar]
  20. Komori T., Okada A., Stewart V., Alt F. W. Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. Science. 1993 Aug 27;261(5125):1171–1175. doi: 10.1126/science.8356451. [DOI] [PubMed] [Google Scholar]
  21. Landau N. R., Schatz D. G., Rosa M., Baltimore D. Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector. Mol Cell Biol. 1987 Sep;7(9):3237–3243. doi: 10.1128/mcb.7.9.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lees-Miller S. P., Godbout R., Chan D. W., Weinfeld M., Day R. S., 3rd, Barron G. M., Allalunis-Turner J. Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line. Science. 1995 Feb 24;267(5201):1183–1185. doi: 10.1126/science.7855602. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Li Z., Otevrel T., Gao Y., Cheng H. L., Seed B., Stamato T. D., Taccioli G. E., Alt F. W. The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell. 1995 Dec 29;83(7):1079–1089. doi: 10.1016/0092-8674(95)90135-3. [DOI] [PubMed] [Google Scholar]
  27. Lieber M. R., Hesse J. E., Lewis S., Bosma G. C., Rosenberg N., Mizuuchi K., Bosma M. J., Gellert M. The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell. 1988 Oct 7;55(1):7–16. doi: 10.1016/0092-8674(88)90004-9. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Mizushima S., Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990 Sep 11;18(17):5322–5322. doi: 10.1093/nar/18.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nussenzweig A., Chen C., da Costa Soares V., Sanchez M., Sokol K., Nussenzweig M. C., Li G. C. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature. 1996 Aug 8;382(6591):551–555. doi: 10.1038/382551a0. [DOI] [PubMed] [Google Scholar]
  31. Oettinger M. A., Schatz D. G., Gorka C., Baltimore D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science. 1990 Jun 22;248(4962):1517–1523. doi: 10.1126/science.2360047. [DOI] [PubMed] [Google Scholar]
  32. Pear W. S., Nolan G. P., Scott M. L., Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. doi: 10.1073/pnas.90.18.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pergola F., Zdzienicka M. Z., Lieber M. R. V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair. Mol Cell Biol. 1993 Jun;13(6):3464–3471. doi: 10.1128/mcb.13.6.3464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Rathmell W. K., Chu G. A DNA end-binding factor involved in double-strand break repair and V(D)J recombination. Mol Cell Biol. 1994 Jul;14(7):4741–4748. doi: 10.1128/mcb.14.7.4741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Roman C. A., Baltimore D. Genetic evidence that the RAG1 protein directly participates in V(D)J recombination through substrate recognition. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2333–2338. doi: 10.1073/pnas.93.6.2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Roth D. B., Lindahl T., Gellert M. Repair and recombination. How to make ends meet. Curr Biol. 1995 May 1;5(5):496–499. doi: 10.1016/s0960-9822(95)00101-1. [DOI] [PubMed] [Google Scholar]
  39. Roth D. B., Menetski J. P., Nakajima P. B., Bosma M. J., Gellert M. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell. 1992 Sep 18;70(6):983–991. doi: 10.1016/0092-8674(92)90248-b. [DOI] [PubMed] [Google Scholar]
  40. Sawchuk D. J., Weis-Garcia F., Malik S., Besmer E., Bustin M., Nussenzweig M. C., Cortes P. V(D)J recombination: modulation of RAG1 and RAG2 cleavage activity on 12/23 substrates by whole cell extract and DNA-bending proteins. J Exp Med. 1997 Jun 2;185(11):2025–2032. doi: 10.1084/jem.185.11.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Schatz D. G., Oettinger M. A., Schlissel M. S. V(D)J recombination: molecular biology and regulation. Annu Rev Immunol. 1992;10:359–383. doi: 10.1146/annurev.iy.10.040192.002043. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Steen S. B., Gomelsky L., Roth D. B. The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo. Genes Cells. 1996 Jun;1(6):543–553. doi: 10.1046/j.1365-2443.1996.d01-259.x. [DOI] [PubMed] [Google Scholar]
  46. Taccioli G. E., Gottlieb T. M., Blunt T., Priestley A., Demengeot J., Mizuta R., Lehmann A. R., Alt F. W., Jackson S. P., Jeggo P. A. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science. 1994 Sep 2;265(5177):1442–1445. doi: 10.1126/science.8073286. [DOI] [PubMed] [Google Scholar]
  47. Taccioli G. E., Rathbun G., Oltz E., Stamato T., Jeggo P. A., Alt F. W. Impairment of V(D)J recombination in double-strand break repair mutants. Science. 1993 Apr 9;260(5105):207–210. doi: 10.1126/science.8469973. [DOI] [PubMed] [Google Scholar]
  48. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Zhu C., Bogue M. A., Lim D. S., Hasty P., Roth D. B. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell. 1996 Aug 9;86(3):379–389. doi: 10.1016/s0092-8674(00)80111-7. [DOI] [PubMed] [Google Scholar]
  51. Zhu C., Roth D. B. Characterization of coding ends in thymocytes of scid mice: implications for the mechanism of V(D)J recombination. Immunity. 1995 Jan;2(1):101–112. doi: 10.1016/1074-7613(95)90082-9. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. van Gent D. C., Mizuuchi K., Gellert M. Similarities between initiation of V(D)J recombination and retroviral integration. Science. 1996 Mar 15;271(5255):1592–1594. doi: 10.1126/science.271.5255.1592. [DOI] [PubMed] [Google Scholar]
  54. 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]

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