Hairpin Coding End Opening Is Mediated by RAG1 and RAG2 Proteins Eva Besmer, Jorge Mansilla-Soto, Sylvanie Cassard, Dennis J Sawchuk, Greg Brown, Moshe Sadofsky, Susanna M Lewis, Michel C Nussenzweig, Patricia Cortes  Molecular Cell  Volume 2, Issue 6, Pages 817-828 (December 1998) DOI: 10.1016/S1097-2765(00)80296-8

Figure 1 Hairpin Nicking by Copurified RAG1/RAG2 (A) DNA sequence of hairpin oligonucleotide DR109, 107, 103, and dumbbell substrate. In all cases, the site of the 32P label is indicated by an asterisk, and the sites of nicking are marked by arrows. The dumbbell double hairpin substrate is a covalently closed DNA circle with an internal 5′ 32P-thymidine and a centrally located EcoRI site. The labeled EcoRI fragment is in italics. The right end of the dumbbell substrate has an 11 bp terminal sequence corresponding to Jk1. (B and C) Hairpin-nicking assays. Copurified RAG1 and RAG2 (RAG1/2) were incubated with substrates labeled at their 5′ ends with T4 kinase (B) or 3′ ends with terminal deoxyribonucleotide transferase (C). Reactions were analyzed on 20% acrylamide urea sequencing gels. Marker lanes (M) for each substrate show the half hairpin product expected from precise cleavage at the hairpin end. The products of hairpin opening are indicated by arrows. (D) Hairpin-nicking reactions with a dumbbell substrate. Dumbbell double hairpin substrate (40 fmol) was incubated with RAGs, digested with EcoRI, and analyzed on 8% polyacrylamide sequencing gels. The length of the products, determined from markers, is indicated. Molecular Cell 1998 2, 817-828DOI: (10.1016/S1097-2765(00)80296-8)

Figure 2 Nicking Requires Both RAG1 and RAG2 Substrate oligonucleotides are diagrammed above each panel. DNA substrates were incubated with RAG1, RAG2, or both, or with copurified RAG1/RAG2 as indicated. Arrows at the side of each panel indicate the size of the products based on comigrating markers of the same sequence. Reactions and gel conditions are as in Figure 1. (A) Hairpin-nicking reaction with DR109 labeled at the 5′ end. MBNa, Mung bean nuclease (2 U) from New England BioLabs; MBNb, Boehringer Mannheim (5 U). (B) Hairpin-nicking reaction with DR109 labeled at the 3′ end by filling in the last base of DR109-T. The dot indicates glycerol adducts to pATOH. (C) Double-stranded 12RSS cleavage with the top strand of the substrate labeled at the 5′ end. (D) Open-ended DR109 nicking with the substrate labeled at the 5′ end of the top strand. (E) DNA-nicking reaction using a single-stranded 5′-labeled 20-mer as substrate. Molecular Cell 1998 2, 817-828DOI: (10.1016/S1097-2765(00)80296-8)

Figure 3 Hairpin-Opening and 3′ End–Processing Activities Copurify with RAG1 and RAG2 Copurified RAGs were fractionated on a Sepharose 200 column. Six μl from each fraction or 2 μl of input RAG1/RAG2 were assayed in (C), (D), and (E). Fraction numbers are given at the top of the figure, and the size and elution of marker molecules used to calibrate the column are indicated in (A) and (B). (A) Silver-stained gel. (B) Western blot, monoclonal anti-RAG1 (top) and polyclonal anti-RAG2 (bottom). (C) Hairpin-nicking reaction with 5′-labeled DR109. (D) Hairpin-nicking reaction with 3′-labeled DR109. The dot indicates glycerol adducts to the dinucleotide product. (E) 12RSS substrate cleavage. The substrate is labeled on the 5′ end of the top strand. Molecular Cell 1998 2, 817-828DOI: (10.1016/S1097-2765(00)80296-8)

Figure 4 RAG1 Mutations and Hairpin Nicking (A) Map of RAG1 mutant constructs. The number of each deletion (Sadofsky et al. 1993) is indicated at the top, and the site of the amino end of each indicated at the bottom. The 127 wild-type control core is indicated in the center of the diagram. (B) Western blot with polyclonal anti-RAG1 and anti-RAG2 antibodies. The number of the RAG mutant is shown above each lane. In 127A, 2.5 μg of RAG1 control (127) and 2.5 μg of RAG2 control (201) were cotransfected. In 127B, 5 μg each of the 127 RAG1 and 201 RAG2 control were used (this amount of DNA was used for all the mutants). (C and D) Hairpin opening and 3′ end processing. Of each RAG1/RAG2 extract, 0.25 to 2 μl was incubated with the DR109 substrate labeled at either the 5′ or 3′ end as indicated. The size of the products is shown. The dot marks glycerol adducts. (E) 12RSS cleavage. The same proteins were incubated with double-stranded 12RSS 5′ labeled on the top strand. The initial cleavage product, 14 nucleotides long, and the hairpin product, 24 bases long, are identified at left. Molecular Cell 1998 2, 817-828DOI: (10.1016/S1097-2765(00)80296-8)

Figure 5 RAG2 Mutations and Hairpin Nicking (A) Map of RAG2 mutations (Sadofsky et al. 1994) annotated as in Figure 4. (B) Western blot as in Figure 4. (C, D, and E) Cleavage assays as in Figures 4C, 4D, 4E. Molecular Cell 1998 2, 817-828DOI: (10.1016/S1097-2765(00)80296-8)

Figure 6 Nicking by RAG1 and RAG2 Can Be Mediated by Alcoholysis Copurified RAG1/RAG2 were incubated with 3′ Klenow-labeled DR109 in reactions containing 20% 1,2 ethanediol or 20% glycerol. Dots indicate alcohol-specific adducts. (A) DR109 nicking and 3′ end processing. Products were resolved on 20% sequencing gels. (B) DR109 nicking and 3′ end processing. Reactions were resolved on 22.5% sequencing gels to visualize the alcohol adducts to the products of hairpin nicking. Molecular Cell 1998 2, 817-828DOI: (10.1016/S1097-2765(00)80296-8)

Figure 7 Hairpin Opening under 12/23-Regulated Conditions Annealed 23RSS oligonucleotides (20 fmol), labeled at the 3′ end of the bottom strand, were incubated with RAGs and 25 ng rHMG in Mg2+. (A) RSS cleavage and hairpin opening using a single RSS. Unlabeled 23RSS (20 fmol) was added to the reaction as indicated. (B) RSS cleavage and hairpin opening using paired 12- and 23RSSs. Unlabeled 12RSS (20 fmol) was added to the reaction as indicated. Reactions were analyzed on 20% sequencing gels. Molecular Cell 1998 2, 817-828DOI: (10.1016/S1097-2765(00)80296-8)