1. The V(D)J Recombinase Efficiently Cleaves and Transposes Signal Joints
Matthew B Neiditch, Gregory S Lee, Leslie E Huye, Vicky L Brandt, David B Roth 
Molecular Cell 
Volume 9, Issue 4, Pages (April 2002)
DOI: /S (02)00494-X

2. Figure 1 Signal Joints Are Cleaved In Vivo
(A) Schematic of an archetypal V(D)J recombination reaction utilizing pJH289 as the recombination substrate. Open and closed triangles represent the 12- and 23-RSS, respectively, in all figures.
(B) Arrow denotes 113 bp LM-PCR product from in vivo recombination reactions. (−) indicates that empty vector was transfected in place of the RAG expression vectors.
(C) Recombination substrates derived from pJH289. Substrates contain a signal joint (pSJ), a 12 RSS only (p12), or a 12/23 RSS pair (pJH289). Dilutions represent the relative amount of ligated DNA that went into the PCR reactions.
Molecular Cell 2002 9, DOI: ( /S (02)00494-X)

3. Figure 2 Mechanisms of Signal Joint Cleavage
(A) Signal joint cleavage could occur via a synaptic complex-dependent mechanism where nick and hairpin formation depend on the presence of a 12- and 23-RSS situated in trans. Step (1), binding of RAG proteins to input substrates forming a synaptic complex in trans; step (2), single-strand nicked intermediates; and step (3), hairpin coding and blunt signal end products.
(B) Synaptic complex-independent signal joint cleavage. Signal joint cleavage could occur via a nick-nick pathway. RAG proteins could cleave a single signal joint by nicking 5′ of each RSS, which could occur either simultaneously or sequentially. Step (1), binding of RAG proteins to input substrates in cis; step (2), single-strand nicked intermediate; and step (3), blunt signal end products. Curved arrows indicate the position of nicking.
Molecular Cell 2002 9, DOI: ( /S (02)00494-X)

4. Figure 3 Signal Joint Cleavage In Vitro Proceeds In cis
Native polyacrylamide gel analysis of in vitro plasmid cleavage reactions. Dialysis buffer was substituted for purified RAG proteins where indicated (−). 208 and 232 bp cleavage products were not resolved from one another under the native gel conditions.
Molecular Cell 2002 9, DOI: ( /S (02)00494-X)

5. Figure 4
Signal Joint Cleavage Occurs Predominantly via a Nick-Nick Mechanism In Vivo and In Vitro
(A) Two-dimensional native (N)/alkaline (D) gel electrophoresis of in vitro signal joint cleavage with purified proteins. For clarity, a lighter exposure of the radiolabeled marker lane is also included.
(B) Two-dimensional native (N)/alkaline (D) gel of in vivo signal joint cleavage. The uncleaved product is 440 nt. The 208 and 232 nt cleavage and nicked products and the 416 and 464 nt hairpinned products were not resolved from one another.
Molecular Cell 2002 9, DOI: ( /S (02)00494-X)

6. Figure 5
Hairpin-Deficient RAG1 Mutants Efficiently Cleave Signal Joints In Vivo
Native polyacrylamide gel electrophoresis of LM-PCR products (arrow) from in vivo transfections of wt RAG1 (+), mutant RAG1 (K890A or R894A), or empty vector (−), and the indicated recombination substrate. Dilutions represent the relative amount of ligated DNA in PCR reactions. All transfections contained wt RAG2 except lanes 4 and 9. All lanes in both the pJH289 and pSJ panels come from the same exposure of the same gels, respectively.
Molecular Cell 2002 9, DOI: ( /S (02)00494-X)

7. Figure 6 Signal Joints Are Good Substrates for Transposition
(A) Schematic of transposition assay with uncleaved or signal joint labeled duplex oligonucleotide donors and unlabeled supercoiled plasmid target. The asterisk indicates the position of the radiolabel.
(B) Transposition products are nicked circular (nc) and linearized plasmid targets (lin). (−) indicates that dialysis buffer was substituted for RAG proteins or annealing buffer was substituted for donor in indicated reactions.
Molecular Cell 2002 9, DOI: ( /S (02)00494-X)