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Nucleic Acids Res. Nov 1, 1997; 25(21): 4370–4378.
PMCID: PMC147051

Nicking is asynchronous and stimulated by synapsis in 12/23 rule-regulated V(D)J cleavage.


The first step in DNA cleavage at V(D)J recombination signals by RAG1 and RAG2 is creation of a nick at the heptamer/coding flank border. Under proper conditions in vitro the second step, hairpin formation, requires two signals with spacers of 12 and 23 bp, a restriction referred to as the 12/23 rule. Under these conditions hairpin formation occurs at the two signals at or near the same time. In contrast, we find that under the same conditions nicking occurs at isolated signals and hence is not subject to the 12/23 rule. With two signals the nicking events are not concerted and the signal with a 12 bp spacer is usually nicked first. However, the extent and rate of nicking at a given signal are diminished by mutations of the other signal. The appearance of DNA nicked at both signals is stimulated by more than an order of magnitude by the ability of the signals to synapse, indicating that synapsis accelerates nicking and often precedes it. These observations allow formulation of a more complete model of catalysis of DNA cleavage and how the 12/23 rule is enforced.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ramsden DA, Baetz K, Wu GE. Conservation of sequence in recombination signal sequence spacers. Nucleic Acids Res. 1994 May 25;22(10):1785–1796. [PMC free article] [PubMed]
  • Hesse JE, Lieber MR, Mizuuchi K, Gellert M. V(D)J recombination: a functional definition of the joining signals. Genes Dev. 1989 Jul;3(7):1053–1061. [PubMed]
  • Lewis SM, Hesse JE. Cutting and closing without recombination in V(D)J joining. EMBO J. 1991 Dec;10(12):3631–3639. [PMC free article] [PubMed]
  • Lewis SM. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv Immunol. 1994;56:27–150. [PubMed]
  • McBlane JF, van Gent DC, Ramsden DA, Romeo C, Cuomo CA, Gellert M, Oettinger MA. 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. [PubMed]
  • van Gent DC, Mizuuchi K, Gellert M. Similarities between initiation of V(D)J recombination and retroviral integration. Science. 1996 Mar 15;271(5255):1592–1594. [PubMed]
  • Roth DB, Menetski JP, Nakajima PB, Bosma MJ, 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. [PubMed]
  • Roth DB, Nakajima PB, Menetski JP, Bosma MJ, 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. [PubMed]
  • Ramsden DA, Gellert M. Formation and resolution of double-strand break intermediates in V(D)J rearrangement. Genes Dev. 1995 Oct 1;9(19):2409–2420. [PubMed]
  • Zhu C, Roth DB. 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. [PubMed]
  • Livák F, Schatz DG. Identification of V(D)J recombination coding end intermediates in normal thymocytes. J Mol Biol. 1997 Mar 21;267(1):1–9. [PubMed]
  • 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. [PubMed]
  • Taccioli GE, Gottlieb TM, Blunt T, Priestley A, Demengeot J, Mizuta R, Lehmann AR, Alt FW, Jackson SP, Jeggo PA. 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. [PubMed]
  • Smider V, Rathmell WK, Lieber MR, Chu G. Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science. 1994 Oct 14;266(5183):288–291. [PubMed]
  • Zhu C, Bogue MA, Lim DS, Hasty P, Roth DB. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell. 1996 Aug 9;86(3):379–389. [PubMed]
  • Kirchgessner CU, Patil CK, Evans JW, Cuomo CA, Fried LM, Carter T, Oettinger MA, Brown JM. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science. 1995 Feb 24;267(5201):1178–1183. [PubMed]
  • Blunt T, Finnie NJ, Taccioli GE, Smith GC, Demengeot J, Gottlieb TM, Mizuta R, Varghese AJ, Alt FW, Jeggo PA, et al. 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. [PubMed]
  • Steen SB, Gomelsky L, Roth DB. 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. [PubMed]
  • Eastman QM, Leu TM, Schatz DG. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature. 1996 Mar 7;380(6569):85–88. [PubMed]
  • van Gent DC, Ramsden DA, 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. [PubMed]
  • Sawchuk DJ, Weis-Garcia F, Malik S, Besmer E, Bustin M, Nussenzweig MC, 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. [PMC free article] [PubMed]
  • van Gent DC, Hiom K, Paull TT, Gellert M. Stimulation of V(D)J cleavage by high mobility group proteins. EMBO J. 1997 May 15;16(10):2665–2670. [PMC free article] [PubMed]
  • Cuomo CA, Mundy CL, Oettinger MA. DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences. Mol Cell Biol. 1996 Oct;16(10):5683–5690. [PMC free article] [PubMed]
  • Ramsden DA, McBlane JF, van Gent DC, 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]
  • Agrawal A, Schatz DG. 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. [PubMed]
  • Hesse JE, Lieber MR, 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. [PubMed]
  • Sheehan KM, Lieber MR. V(D)J recombination: signal and coding joint resolution are uncoupled and depend on parallel synapsis of the sites. Mol Cell Biol. 1993 Mar;13(3):1363–1370. [PMC free article] [PubMed]
  • Thompson CB. New insights into V(D)J recombination and its role in the evolution of the immune system. Immunity. 1995 Nov;3(5):531–539. [PubMed]
  • Lewis SM, Wu GE. The origins of V(D)J recombination. Cell. 1997 Jan 24;88(2):159–162. [PubMed]
  • Sakai J, Chalmers RM, Kleckner N. Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition. EMBO J. 1995 Sep 1;14(17):4374–4383. [PMC free article] [PubMed]
  • Gary PA, Biery MC, Bainton RJ, Craig NL. Multiple DNA processing reactions underlie Tn7 transposition. J Mol Biol. 1996 Mar 29;257(2):301–316. [PubMed]
  • Savilahti H, Mizuuchi K. Mu transpositional recombination: donor DNA cleavage and strand transfer in trans by the Mu transposase. Cell. 1996 Apr 19;85(2):271–280. [PubMed]
  • Bainton R, Gamas P, Craig NL. Tn7 transposition in vitro proceeds through an excised transposon intermediate generated by staggered breaks in DNA. Cell. 1991 May 31;65(5):805–816. [PubMed]
  • Haniford D, Kleckner N. Tn 10 transposition in vivo: temporal separation of cleavages at the two transposon ends and roles of terminal basepairs subsequent to interaction of ends. EMBO J. 1994 Jul 15;13(14):3401–3411. [PMC free article] [PubMed]
  • Baker TA, Luo L. Identification of residues in the Mu transposase essential for catalysis. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6654–6658. [PMC free article] [PubMed]
  • Bolland S, Kleckner N. The three chemical steps of Tn10/IS10 transposition involve repeated utilization of a single active site. Cell. 1996 Jan 26;84(2):223–233. [PubMed]
  • Rice P, Mizuuchi K. Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration. Cell. 1995 Jul 28;82(2):209–220. [PubMed]
  • Sadofsky MJ, Hesse JE, van Gent DC, 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. [PubMed]
  • Rodgers KK, Bu Z, Fleming KG, Schatz DG, Engelman DM, Coleman JE. A zinc-binding domain involved in the dimerization of RAG1. J Mol Biol. 1996 Jul 5;260(1):70–84. [PubMed]
  • Difilippantonio MJ, McMahan CJ, Eastman QM, Spanopoulou E, Schatz DG. RAG1 mediates signal sequence recognition and recruitment of RAG2 in V(D)J recombination. Cell. 1996 Oct 18;87(2):253–262. [PubMed]
  • Spanopoulou E, Zaitseva F, Wang FH, 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. [PubMed]
  • 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. [PubMed]

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