1. Volume 7, Issue 1, Pages 65-75 (January 2001)
Joining-Deficient RAG1 Mutants Block V(D)J Recombination In Vivo and Hairpin Opening In Vitro 
Heather Yarnall Schultz, Mark A. Landree, Jian-xia Qiu, Sam B. Kale, David B. Roth 
Molecular Cell 
Volume 7, Issue 1, Pages (January 2001)
DOI: /S (01)

2. Figure 1
Effects of RAG1 Mutations on Protein Expression, Cleavage, and Joining In Vivo
(A) Western blot of RAG proteins in cell lysates from transient transfections. Separated proteins were probed with anti-c-myc antibodies and HRP-conjugated anti-mouse IgG1 antibodies and visualized with the ECL-PLUS kit. The asterisk denotes a degradation product that is occasionally seen with both mutants and wild-type RAG1 proteins.
(B) In vivo analysis of coding joints. One three-hundredth of a transfection was used in a PCR reaction for coding joint formation using the DR99 and ML68 primers. Transfections of wild-type RAG proteins were assayed at 1×, 1:10, and 1:100 dilutions; mutants and mock transfections lacking RAG expression vectors (“no RAGs”) were assayed without dilution. Reactions were run on polyacrylamide gels, blotted, and probed with DR99. All lanes are from the same gel. Squares indicate coding segments; arrows indicate PCR primers.
(C) In vivo analysis of signal joints. One three-hundredth of a transfection was used in a PCR reaction for signal joint formation using the DR55 and DR100 primers. The blot was probed with DR55. Open triangle indicates the 12-RSS; closed triangle denotes the 23-RSS.
(D and E) Southern blot analysis of E547Q and E423Q. For each mutant, two independent transfections with wild-type RAG2 were performed. Five-sixths of the DNA harvested from a transfection was electrophoresed through an agarose gel without restriction digestion to allow visualization of the doubly cleaved signal end fragment from pJH290 (D) or the doubly cleaved coding end fragment from pJH289 (E). The unrearranged substrate and cleaved plasmid backbone fragments do not appear because these large molecules transfer poorly under the conditions used. The blot was probed with a radiolabeled PvuII fragment of the pJH290 that contains the unrearranged signal pair and spans the entire length of the excised fragment.
Molecular Cell 2001 7, 65-75DOI: ( /S (01) )

3. Figure 2 Nucleotide Sequence Analysis of Coding Joints
Nucleotide sequences of junctions formed from multiple transfections of two different substrates, pJH290 (deletional recombination) and pJH299 (inversional recombination) are shown. (A), junctions formed by E547Q; (B), junctions formed by E423Q. Similar results were obtained with both substrates, so the data are shown together. The sequence of a perfect coding joint is shown at the top. The number of nucleotides deleted from each coding end is given for each sequence. Capital letters indicate P nucleotides. Lowercase letters indicate N nucleotides. The number of junctions isolated with the indicated sequence is given in parentheses. “H” indicates joints containing short sequence homologies; “+TdT” indicates the junctions were isolated from transfections in which terminal deoxynucleotidyl transferase was present.
Molecular Cell 2001 7, 65-75DOI: ( /S (01) )

4. Figure 3 Nicking and Hairpin Formation by Purified Proteins
Cleavage activity was measured using GST-fusion proteins copurified from transfections with either wild-type or mutant GST-RAG1 and wild-type GST-RAG2. The 12-RSS oligonucleotide substrate (DAR39/40) was radiolabeled at the 5′ end of the top strand. Cleavage produces a 16 nucleotide nicked product and a 32 nucleotide hairpin product.
(A) Cleavage at the 12 RSS was assayed in 5 mM MnCl2.
(B) Coupled cleavage was assayed in 5 mM MgCl2 in the presence of an unlabeled oligonucleotide containing a 23-RSS (DG61/62). Equivalent amounts of RAG proteins (as determined by Coomassie- stained SDS–PAGE) were used in each assay.
Molecular Cell 2001 7, 65-75DOI: ( /S (01) )

5. Figure 4 Efficient Hybrid Joint Formation by Both E547Q and E423Q
(A) Schematic of hybrid joint formation with pJH299.
(B) Hybrid joints formed from the pJH299 substrate in vitro were measured by a PCR assay that detects hybrid joints formed on the excised circle. RAG GST-fusion proteins were incubated with the plasmid substrate. One-fifth of the reaction mix was assayed by PCR using the DR55 and ML68 primers. The blot was probed with the joint-specific DR98 oligonucleotide. Symbols are as in Figure 1. All lanes are from the same gel.
Molecular Cell 2001 7, 65-75DOI: ( /S (01) )

6. Figure 5 Hairpin Opening Is Impaired by E423Q
(A) A 5′ end-labeled hairpin oligonucleotide (DR109) was used as a substrate. GST-fusion proteins were incubated with this substrate in 10 mM MnCl2 at 30°C for 90 min. Hairpin opening immediately 5′ of the hairpin tip liberates 18- and 17-nucleotide products. Products resulting from cleavage near the 3′ end of the substrate (3′ end processing) are also observed.
(B) Kinetic analysis of hairpin-opening activity of E547Q and E423Q. Reactions were performed as in (A) except that they were terminated at 0, 15, 30, 60, 90, or 180 min. Equivalent amounts of RAG proteins (as determined by Coomassie-stained SDS–PAGE) were used in each assay. All lanes are from the same gel.
Molecular Cell 2001 7, 65-75DOI: ( /S (01) )

7. Figure 6 Both E547Q and E423Q Are Capable of Efficient Transposition
(A) Schematic of the in vitro transposition reaction.
(B) RAG GST-fusion proteins were incubated at 37°C with labeled 12 and 23 RSS in the presence of CaCl2. Plasmid target and MgCl2 were added, and the reactions were again incubated at 37°C. Products were separated on a 4%–20% gradient gel. Nicked products indicate single-ended transposition events, and linear products indicate double-ended transposition events.
Molecular Cell 2001 7, 65-75DOI: ( /S (01) )