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. 1998 Aug 15;26(16):3769–3775. doi: 10.1093/nar/26.16.3769

V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells.

J O Han 1, L A Erskine 1, M M Purugganan 1, T D Stamato 1, D B Roth 1
PMCID: PMC147771  PMID: 9685494

Abstract

V(D)J recombination assembles immunoglobulin (Ig) and T cell receptor (TCR) gene segments during lymphocyte development. Recombination is initiated by the RAG-1 and RAG-2 proteins, which introduce double-stranded DNA breaks (DSB) adjacent to the Ig and TCR gene segments. The broken ends are joined by the DSB repair machinery, which includes the XRCC4 protein. While XRCC4 is essential for both DSB repair and V(D)J recombination, the functions of this protein remain enigmatic. Because the rare V(D)J recombination products isolated from XRCC4-deficient cells generally show evidence of excessive nucleotide loss, it was hypothesized that XRCC4 may function to protect broken DNA ends. Here we report the first examination of V(D)J recombination intermediates in XRCC4-deficient cells. We found that both types of intermediates, signal ends and coding ends, are abundant in the absence of XRCC4. Furthermore, the signal ends are full length. We also showed that alternative V(D)J recombination products, hybrid joints, form with normal efficiency and without excessive deletion in XRCC4-deficient cells. These data indicate that impaired formation of V(D)J recombination products in XRCC4-deficient cells does not result from excessive degradation of recombination intermediates. Potential roles of XRCC4 in the joining reaction are discussed.

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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. Bogue M. A., Wang C., Zhu C., Roth D. B. V(D)J recombination in Ku86-deficient mice: distinct effects on coding, signal, and hybrid joint formation. Immunity. 1997 Jul;7(1):37–47. doi: 10.1016/s1074-7613(00)80508-7. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Critchlow S. E., Bowater R. P., Jackson S. P. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol. 1997 Aug 1;7(8):588–598. doi: 10.1016/s0960-9822(06)00258-2. [DOI] [PubMed] [Google Scholar]
  6. Getts R. C., Stamato T. D. Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J Biol Chem. 1994 Jun 10;269(23):15981–15984. [PubMed] [Google Scholar]
  7. Grawunder U., Wilm M., Wu X., Kulesza P., Wilson T. E., Mann M., Lieber M. R. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature. 1997 Jul 31;388(6641):492–495. doi: 10.1038/41358. [DOI] [PubMed] [Google Scholar]
  8. Han J. O., Steen S. B., Roth D. B. Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products. Mol Cell Biol. 1997 Apr;17(4):2226–2234. doi: 10.1128/mcb.17.4.2226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Jeggo P. A. Studies on mammalian mutants defective in rejoining double-strand breaks in DNA. Mutat Res. 1990 Jul;239(1):1–16. doi: 10.1016/0165-1110(90)90028-a. [DOI] [PubMed] [Google Scholar]
  11. Jeggo P. A. X-ray sensitive mutants of Chinese hamster ovary cell line: radio-sensitivity of DNA synthesis. Mutat Res. 1985 May;145(3):171–176. doi: 10.1016/0167-8817(85)90024-0. [DOI] [PubMed] [Google Scholar]
  12. Kao F., Chasin L., Puck T. T. Genetics of somatic mammalian cells. X. Complementation analysis of glycine-requiring mutants. Proc Natl Acad Sci U S A. 1969 Dec;64(4):1284–1291. doi: 10.1073/pnas.64.4.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lafaille J. J., DeCloux A., Bonneville M., Takagaki Y., Tonegawa S. Junctional sequences of T cell receptor gamma delta genes: implications for gamma delta T cell lineages and for a novel intermediate of V-(D)-J joining. Cell. 1989 Dec 1;59(5):859–870. doi: 10.1016/0092-8674(89)90609-0. [DOI] [PubMed] [Google Scholar]
  14. Leber R., Wise T. W., Mizuta R., Meek K. The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase. J Biol Chem. 1998 Jan 16;273(3):1794–1801. doi: 10.1074/jbc.273.3.1794. [DOI] [PubMed] [Google Scholar]
  15. Lewis S. M., Hesse J. E., Mizuuchi K., Gellert M. Novel strand exchanges in V(D)J recombination. Cell. 1988 Dec 23;55(6):1099–1107. doi: 10.1016/0092-8674(88)90254-1. [DOI] [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. Lewis S., Gellert M. The mechanism of antigen receptor gene assembly. Cell. 1989 Nov 17;59(4):585–588. doi: 10.1016/0092-8674(89)90002-0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  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. Meier J. T., Lewis S. M. P nucleotides in V(D)J recombination: a fine-structure analysis. Mol Cell Biol. 1993 Feb;13(2):1078–1092. doi: 10.1128/mcb.13.2.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Melek M., Gellert M., van Gent D. C. Rejoining of DNA by the RAG1 and RAG2 proteins. Science. 1998 Apr 10;280(5361):301–303. doi: 10.1126/science.280.5361.301. [DOI] [PubMed] [Google Scholar]
  22. Merrihew R. V., Sargent R. G., Wilson J. H. Efficient modification of the APRT gene by FLP/FRT site-specific targeting. Somat Cell Mol Genet. 1995 Sep;21(5):299–307. doi: 10.1007/BF02257465. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Ramsden D. A., Gellert M. Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks. EMBO J. 1998 Jan 15;17(2):609–614. doi: 10.1093/emboj/17.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Roth D. B., Nakajima P. B., Menetski J. P., Bosma M. J., Gellert M. V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals. Cell. 1992 Apr 3;69(1):41–53. doi: 10.1016/0092-8674(92)90117-u. [DOI] [PubMed] [Google Scholar]
  29. Roth D. B., Zhu C., Gellert M. Characterization of broken DNA molecules associated with V(D)J recombination. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10788–10792. doi: 10.1073/pnas.90.22.10788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sadofsky M. J., Hesse J. E., Gellert M. Definition of a core region of RAG-2 that is functional in V(D)J recombination. Nucleic Acids Res. 1994 May 25;22(10):1805–1809. doi: 10.1093/nar/22.10.1805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Schlissel M., Constantinescu A., Morrow T., Baxter M., Peng A. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 1993 Dec;7(12B):2520–2532. doi: 10.1101/gad.7.12b.2520. [DOI] [PubMed] [Google Scholar]
  33. Stamato T. D., Weinstein R., Giaccia A., Mackenzie L. Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell. Somatic Cell Genet. 1983 Mar;9(2):165–173. doi: 10.1007/BF01543175. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Steen S. B., Gomelsky L., Speidel S. L., Roth D. B. Initiation of V(D)J recombination in vivo: role of recombination signal sequences in formation of single and paired double-strand breaks. EMBO J. 1997 May 15;16(10):2656–2664. doi: 10.1093/emboj/16.10.2656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. 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]

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