V(D)J Recombination and RAG-Mediated Transposition in Yeast Anne E. Clatworthy, Maria A. Valencia, James E. Haber, Marjorie A. Oettinger  Molecular Cell  Volume 12, Issue 2, Pages 489-499 (August 2003) DOI: 10.1016/S1097-2765(03)00305-8

Figure 1 Reconstitution of V(D)J Reactions in Yeast with RAG1, RAG2, and a Recombination Substrate (A) Schematic diagrams of high copy plasmids used to transform yeast. Substrate plasmid pACY16 contains a 12RSS (white triangle) and 23RSS (black triangle) downstream of the yeast actin promoter. The plasmids present in strains ACY159 and ACY160 are as shown, with ACY159 expressing core versions of RAG1 and RAG2 (RAG1c, RAG2c) and ACY160 expressing their full-length counterparts RAG1FL and RAG2FL. (B and C) Time course of expression of core RAG1/2 in strain ACY159 and full-length RAG1/2 in strain ACY160 following galactose induction. The position of the proteins as detected by immunoblot analysis with (B) anti-hemagglutinin antibody (RAG1) and (C) anti-RAG2 antibody is shown. Molecular Cell 2003 12, 489-499DOI: (10.1016/S1097-2765(03)00305-8)

Figure 2 Nested PCR Detects Signal Joints and Intramolecular Transposition Products in Yeast (A) (Left) Signal ends can be fused to form a signal joint circle. (Middle) A RAG-mediated intramolecular transposition reaction in which the 3′ hydroxyl groups attack the strand opposite to the one from which they derive will create an inversion circle, with RSSs flanked by target site duplications. (Right) A RAG-mediated intramolecular transposition in which the 3′ hydroxyl groups attack the same strand from which they derive will create two deletion circles, only one of which is recoverable by nested PCR. Triangles indicate the location of RSSs and vertical lines the insertion sites (and position of target site duplications). The locations of nested PCR primers used to detect signal joints and transposition products are marked by straight arrows, and the distance between these primers and of the entire signal-end product is shown. Hairpinned coding ends and coding joints were not assayed for. (B) Nested PCR products of the size expected for signal joints and inversion circles (∼536 bp, 968–432 bp) and products of the size expected for deletion circles (<536 bp) are detectable only in yeast strain ACY159 induced to express the RAG proteins (lanes 17–24) and not in controls (lanes 3–16). Lane 1 is a 100 bp DNA ladder. Molecular Cell 2003 12, 489-499DOI: (10.1016/S1097-2765(03)00305-8)

Figure 3 Frequency and Characterization of Intramolecular Transposition Products and Signal Joints (A) Recovery of intramolecular transposition products and signal joints. Transposition products are observed more frequently than signal joints in strains expressing the core RAG proteins (ACY142 and ACY159). Both transposition and signal joining products are detected in strains expressing the full-length RAG proteins (ACY160), although at a greatly reduced frequency. Transposition products are recovered but signal joints are not recovered in lif1Δ strains (ACY174 and ACY175). (B) Target site duplications observed in inversion circles recovered from strains expressing either core RAG1/2 (ACY142 and ACY159) or full-length RAG1/2 (ACY160). The duplications are 4 and 5 bp in length and are predominantly GC rich. “N” indicates the number of insertions observed into a single target site. * indicates that insertion into the target site was observed in multiple inductions. (C) Distribution of intramolecular transposition target sites along the length of the 968 bp signal-ended product observed in strains expressing core RAG1/2 (ACY159 and ACY142). The numbers above or flanking symbols indicate multiple events occurring at a single position. Molecular Cell 2003 12, 489-499DOI: (10.1016/S1097-2765(03)00305-8)

Figure 4 Repair of Deletion but Not Inversion Circles (A) Schematic diagram of S1 sensitivity assay. Signal joints and intramolecular transposition products that have been repaired are insensitive to S1 nuclease and are amplifiable by nested PCR. Transposition products that are not repaired and remain single-stranded at the target site duplications are sensitive to treatment with S1 nuclease and then cannot be amplified by PCR. (B) Inversion circles are sensitive to treatment with S1 nuclease while deletion circles are not. The total number of PCR reactions conducted on DNA from strains “wild-type” (WT) for NHEJ activity (ACY142 and ACY159) and lif1Δ strains (ACY174 and ACY175) is shown. The presence (+) or absence (−) of S1 is indicated. SJs, signal joints; INV, inversion circles; DEL, deletion circles. Molecular Cell 2003 12, 489-499DOI: (10.1016/S1097-2765(03)00305-8)