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. 1997 May 15;16(10):2665–2670. doi: 10.1093/emboj/16.10.2665

Stimulation of V(D)J cleavage by high mobility group proteins.

D C van Gent 1, K Hiom 1, T T Paull 1, M Gellert 1
PMCID: PMC1169877  PMID: 9184213

Abstract

V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 bp between the conserved heptamer and nonamer elements. The RAG1 and RAG2 proteins initiate the reaction by making double-strand DNA breaks at both signals, and must thus be able to operate on these two different spatial arrangements. We show that the DNA-bending proteins HMG1 and HMG2 stimulate cleavage and RAG protein binding at the 23 bp spacer signal. These findings suggest that DNA bending is important for bridging the longer spacer, and explain how a similar array of RAG proteins could accommodate a signal with either a 12 or a 23 bp spacer. An additional effect of HMG proteins is to stimulate coupled cleavage greatly when both signal sequences are present, suggesting that these proteins also aid the formation of a synaptic complex.

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Selected References

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  1. Chen Y. Y., Wang L. C., Huang M. S., Rosenberg N. An active v-abl protein tyrosine kinase blocks immunoglobulin light-chain gene rearrangement. Genes Dev. 1994 Mar 15;8(6):688–697. doi: 10.1101/gad.8.6.688. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Gellert M. Molecular analysis of V(D)J recombination. Annu Rev Genet. 1992;26:425–446. doi: 10.1146/annurev.ge.26.120192.002233. [DOI] [PubMed] [Google Scholar]
  4. Goodman S. D., Nash H. A. Functional replacement of a protein-induced bend in a DNA recombination site. Nature. 1989 Sep 21;341(6239):251–254. doi: 10.1038/341251a0. [DOI] [PubMed] [Google Scholar]
  5. Grosschedl R., Giese K., Pagel J. HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 1994 Mar;10(3):94–100. doi: 10.1016/0168-9525(94)90232-1. [DOI] [PubMed] [Google Scholar]
  6. Haykinson M. J., Johnson R. C. DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly. EMBO J. 1993 Jun;12(6):2503–2512. doi: 10.1002/j.1460-2075.1993.tb05905.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Javaherian K., Liu J. F., Wang J. C. Nonhistone proteins HMG1 and HMG2 change the DNA helical structure. Science. 1978 Mar 24;199(4335):1345–1346. doi: 10.1126/science.628842. [DOI] [PubMed] [Google Scholar]
  9. Lavoie B. D., Chaconas G. A second high affinity HU binding site in the phage Mu transpososome. J Biol Chem. 1994 Jun 3;269(22):15571–15576. [PubMed] [Google Scholar]
  10. Lavoie B. D., Chaconas G. Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes. J Biol Chem. 1990 Jan 25;265(3):1623–1627. [PubMed] [Google Scholar]
  11. Lavoie B. D., Shaw G. S., Millner A., Chaconas G. Anatomy of a flexer-DNA complex inside a higher-order transposition intermediate. Cell. 1996 May 31;85(5):761–771. doi: 10.1016/s0092-8674(00)81241-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Mizuuchi K. Transpositional recombination: mechanistic insights from studies of mu and other elements. Annu Rev Biochem. 1992;61:1011–1051. doi: 10.1146/annurev.bi.61.070192.005051. [DOI] [PubMed] [Google Scholar]
  14. Paull T. T., Carey M., Johnson R. C. Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro. Genes Dev. 1996 Nov 1;10(21):2769–2781. doi: 10.1101/gad.10.21.2769. [DOI] [PubMed] [Google Scholar]
  15. Paull T. T., Haykinson M. J., Johnson R. C. The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures. Genes Dev. 1993 Aug;7(8):1521–1534. doi: 10.1101/gad.7.8.1521. [DOI] [PubMed] [Google Scholar]
  16. Paull T. T., Johnson R. C. DNA looping by Saccharomyces cerevisiae high mobility group proteins NHP6A/B. Consequences for nucleoprotein complex assembly and chromatin condensation. J Biol Chem. 1995 Apr 14;270(15):8744–8754. doi: 10.1074/jbc.270.15.8744. [DOI] [PubMed] [Google Scholar]
  17. Pil P. M., Chow C. S., Lippard S. J. High-mobility-group 1 protein mediates DNA bending as determined by ring closures. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9465–9469. doi: 10.1073/pnas.90.20.9465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Rice P. A., Yang S., Mizuuchi K., Nash H. A. Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell. 1996 Dec 27;87(7):1295–1306. doi: 10.1016/s0092-8674(00)81824-3. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Sadofsky M. J., Hesse J. E., van Gent D. C., Gellert M. RAG-1 mutations that affect the target specificity of V(D)j recombination: a possible direct role of RAG-1 in site recognition. Genes Dev. 1995 Sep 1;9(17):2193–2199. doi: 10.1101/gad.9.17.2193. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Segall A. M., Goodman S. D., Nash H. A. Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins. EMBO J. 1994 Oct 3;13(19):4536–4548. doi: 10.1002/j.1460-2075.1994.tb06775.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shykind B. M., Kim J., Sharp P. A. Activation of the TFIID-TFIIA complex with HMG-2. Genes Dev. 1995 Jun 1;9(11):1354–1365. doi: 10.1101/gad.9.11.1354. [DOI] [PubMed] [Google Scholar]
  27. Singh J., Dixon G. H. High mobility group proteins 1 and 2 function as general class II transcription factors. Biochemistry. 1990 Jul 3;29(26):6295–6302. doi: 10.1021/bi00478a026. [DOI] [PubMed] [Google Scholar]
  28. Slominski A., Ermak G., Hwang J., Mazurkiewicz J., Corliss D., Eastman A. The expression of proopiomelanocortin (POMC) and of corticotropin releasing hormone receptor (CRH-R) genes in mouse skin. Biochim Biophys Acta. 1996 Mar 15;1289(2):247–251. doi: 10.1016/0304-4165(95)00159-x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Stros M., Stokrová J., Thomas J. O. DNA looping by the HMG-box domains of HMG1 and modulation of DNA binding by the acidic C-terminal domain. Nucleic Acids Res. 1994 Mar 25;22(6):1044–1051. doi: 10.1093/nar/22.6.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Werner M. H., Huth J. R., Gronenborn A. M., Clore G. M. Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex. Cell. 1995 Jun 2;81(5):705–714. doi: 10.1016/0092-8674(95)90532-4. [DOI] [PubMed] [Google Scholar]
  32. Yang S. W., Nash H. A. Comparison of protein binding to DNA in vivo and in vitro: defining an effective intracellular target. EMBO J. 1995 Dec 15;14(24):6292–6300. doi: 10.1002/j.1460-2075.1995.tb00319.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  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., 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]
  36. 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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