Targeted transposition by the V(D)J recombinase

Abstract

Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by--and specifically targeted to--hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected.

FIG. 1.

Preferential transposition into an inverted repeat. (A) Schematic of transposition into plasmid targets. Oligonucleotide donors contained either a 12 RSS (open triangles) or a 23 RSS (filled triangles). The 12 RSS-containing oligonucleotide was ³²P end labeled (asterisk). Transposition into random sites followed by ScaI digestion generates products of many different sizes. Transposition into a single hot spot, however, followed by ScaI digestion yields two distinct products. (B) Transposition reactions were carried out as described in the text, with either no target or 0.11 pmol of F14C or pUC8 plasmid target. MnCl₂ (3 mM) was used as the divalent metal ion. (lin, linearized target DNA; dig, products resulting from ScaI digestion). (C) Transposition reactions were carried out as for panel B, with 3 mM MgCl₂.

FIG. 2.

Inverted repeats stimulate transposition. Transposition reactions were carried out as for Fig. 1B, without ScaI digestion. The target consisted of either a single plasmid or a mixture of F14C and pCDNA1-amp in the indicated mass ratios. For mixtures containing a mixture of targets, the preference for F14C was calculated as the amount of linearized F14C divided by the amount of linearized pCDNA1-amp. All lanes are from the same gel.

FIG. 3.

Cruciform structures are hot spots for transposition. (A) F14C was treated with topoisomerase as described in the text. Topoisomers were used as targets for transposition as described for Fig. 1B. Products were resolved on a 1% alkaline agarose gel, dried, and visualized by autoradiography. (B) Plasmids containing various cruciform-forming inverted repeats were used as targets and also show targeting of transposition.

FIG. 4.

Transposition is targeted to the hairpin ends of cruciform arms. (A) Products of transposition into F14C were PCR cloned and sequenced. Sites of integration are marked (∗, clones sequenced from the 12 RSS; +; clones sequenced from the 23 RSS). (B) Products of transposition into the 21-bp stem used in Fig. 3B were sequenced as for panel A. (C) Schematic of integration into a cruciform end, followed by digestion with EcoRI. For clarity, the 23 RSS donor and the complementary strand are not shown. (D) Products of transposition into F14C were digested with EcoRI and run on a denaturing 6% acrylamide gel. A single product is formed, indicating transposition into a single site. The top of the gel was removed for layout purposes; all bands present on the entire gel are visible in this figure.

FIG. 5.

Transposition is targeted to hairpins. (A) Transposition was carried out as described in the text. “Precleaved donor” refers to an oligonucleotide donor that corresponds to the RSS after cleavage, i.e., the donor terminates at the site of cleavage (19). The double-stranded control oligonucleotide was FM116/117, and the hairpin oligonucleotide was HY9. Some products of transposition into free donor molecules are visible in the absence of target. The nonspecific target shows no unique bands, while the hairpin target shows several products which correspond to transposition into the hairpin end. (B) The formation of target capture complexes (TCC) was assayed with double-stranded (MM30) and hairpin (HY9) oligonucleotides. HY9 forms substantially more target capture complexes than the MM30 control. The nonspecific shift of target in the absence of donor is indicated (NS). All lanes are from the same gel.

FIG. 6.

A cruciform-containing plasmid specifically inhibits hybrid joint formation. (A) Schematic of the hybrid joining assay. A plasmid substrate, pJH299, is incubated with RAG proteins. Hybrid joints are detected by PCR with primers at the indicated sites. (B) The presence of F14C inhibits formation of hybrid joints in this assay, while the presence of pUC8 control has little or no effect.