Double-Strand Break Formation by the RAG Complex at the Bcl-2 Major Breakpoint Region and at Other Non-B DNA Structures In Vitro

RAG cleavage sites with respect to the location of the structure formation at the bcl-2 Mbr. In all panels, the 150-bp Mbr is depicted and peaks I, II, and III are indicated by short horizontal lines between the top and bottom strands (represented by the long lines). The arrows pointing downward refer to RAG nicks on the top strand, and arrows pointing upward refer to RAG nicks on the bottom strand. The positions of RAG cleavage shown are determined on the basis of results obtained from both native and denaturing PAGE. Dashed boxes indicate combinations of top and bottom strand nicks that are most likely responsible for the double-strand breaks seen on the corresponding native gels in Fig. 1. A. RAG cleavage on a 248-bp Mbr-containing fragment. Here, the non-B structure formation is upstream of peak I and is represented as the nonduplex region. The actual nature of the non-B structure is not assumed here, but it is not the simple bubble depicted; this is just the simplest DNA deviation for illustrative purposes. The numbered arrows correspond to the numbered cleavage bands in Fig. S2 of the supplemental material. B. RAG cleavage on a 339-bp Mbr-containing fragment. Here, the structure formation is downstream of peak I and peak III. The numbered arrows correspond to the numbered cleavage bands in Fig. S3 in the supplemental material. C. Sites of RAG nicking on plasmid DNA containing the bcl-2 Mbr. Here, RAG nicking sites are detected by primer extension, indicated by open arrows. See the legend of Fig. S4 in the supplemental material for additional details.

RAG cleavage on a 3-bp bcl-2 Mbr mutant. A. The Mbr portion of the 248-bp fragment containing a CCC↔ GGG exchange between the top strand and bottom strands is shown. Note that the Mbr portion is only 150 bp and, hence, the 248-bp fragment is longer than shown in the diagram. The position of the 3-nt exchange is indicated by “xxx.” The region of structure formation is marked as in Fig. 2A. For other details, refer to Fig. 2. B. The 248-bp PCR fragment containing the mutated bcl-2 Mbr or the wild-type Mbr was then end-labeled and studied in the presence or absence of the RAG complex. RAG incubation products were then loaded onto a native PAGE. Lane M is the marker. The positions of the duplex DNA and shifted band containing the non-B DNA are indicated. The new band generated by RAG cleavage is indicated by an arrow. Each sample was prepared in duplicate and loaded in duplicate lanes. Both panels of the figure are derived from the same gel.

Comparison of core, full-length, and mutant RAG-induced DSB formation at the bcl-2 Mbr. A 248-bp (A) or 339-bp (B) DNA fragment of the bcl-2 Mbr containing non-B structure was incubated with different RAG preparations (see below). The products were resolved on a 6% PAGE. (See Fig. 1 legend for details.) In both panels A and B, the descriptions of the lanes are as follows: RAG-induced DSB formation with no RAG protein (lane 1); core GST-RAGs (stands for core GST-RAG1/core GST-RAG2) (lane 2); core MBP-RAGs (stands for core MBP-RAG1/core MBP-RAG2) (lane 3); mutant RAGs (stands for mutant RAG1/core MBP-RAG2) (lane 4); full-length RAGs (stands for core MBP-RAG1/full-length MBP-RAG2) (lane 5). The RAG-sensitive regions are indicated by arrows. Lane M, molecular weight markers. The faint band at 210 bp in panel A is present in all lanes.

RAG cleavage on a DNA bubble and a heterologous loop. Increasing concentrations of RAG1 and RAG2 complexes were incubated with a [γ-³²P]ATP end-labeled 50-bp ds oligomers containing a symmetric bubble structure (A) or a heterologous loop structure (B) for 1 h at 37°C in buffer A. Afterwards, the cleavage products were resolved on a 15% denaturing polyacrylamide gel. In both panels A and B, the lanes are as follows: 1, 50-bp DNA containing the DNA treated with 0 ng RAGs; lane 2, 50 ng RAGs; lane 3, 100 ng RAGs; lane M, 50-bp ladder. In both panels, the sites of RAG cleavage with respect to the corresponding structure are indicated by long arrows. Corresponding nucleotide positions in the structure are marked. The [γ-³²P]ATP-labeled 5′ end of the structure is indicated with an asterisk. Intervening lanes between the marker lane and lanes 1 to 3 have been removed. The band in panel B, lane 3, at ∼32 bp is obscured somewhat by a diffuse band that is present across the gel (even in the blank lane between lanes 1 and 2) just below that position.

Comparison of RAG cleavage on different symmetric bubble structures versus double- and single-stranded DNA substrates. RAG complexes (100 ng) were incubated with a [γ-³²P]ATP end-labeled 50-bp double-stranded DNA, 50-nt single-stranded DNA, or a symmetric bubble structure (see bottom of gel) for 1 h at 37°C in buffer A. The cleavage products were resolved on a 15% denaturing polyacrylamide gel. Two different oligomers, SCR174 (lanes 1 and 2) or YM21 (lanes 3 and 4) were used. A 50-bp double-stranded DNA was prepared by annealing YM21 and SCR 174 (lanes 5 and 6). Symmetric bubbles were prepared as described in Materials and Methods (lanes 7 to 10). The position of the bubble is indicated by the bracket. Symmetric bubbles containing G-C clamps were prepared by annealing SCR175 and SCR176 (lanes 11 and 12). Bubbles with A-T regions were prepared by annealing SCR177 and SCR178 (lanes 13 and 14). In all cases except lanes 9 and 10, core GST-RAG proteins were used. In the case of lane 9, core MBP-RAG1 and core MBP-RAG2 were used. For lane 10, the core MBP-RAG1 active site mutant and core MBP-RAG2 were used.

RAGs induce double-strand breaks on symmetric bubble and heterologous loop structures. A. Gel showing RAG-induced nicks on both strands of the symmetric bubble and heterologous loop structures. Core GST-RAG proteins (100 ng) were incubated with a [γ-³²P]ATP end-labeled 50-bp symmetric bubble structure or heterologous loop structure for 1 h at 37°C. Afterwards, the cleavage products were resolved on a 15% denaturing polyacrylamide gel. In order to detect the RAG nicking at the top or bottom strand, the respective strand of the heterologous loop or symmetric bubble structure was [γ-³²P]ATP end-labeled and used for RAG cleavage. M and M′ stand for molecular weight markers. The bubble region of the symmetric bubble is demarcated. The loop region of the top strand of the heterologous loop is demarcated; note that this does not apply to the bottom strand. RAG nicking positions are indicted by arrows. B. Schematic presentation of RAG nicking positions on symmetric bubble and heterologous loop structures. Oligomers used for making the structures are indicated. RAG nicking positions on both strands are indicted by an arrowhead. The asterisk next to the arrowhead indicates that this cleavage is noted on a different gel (Fig. 5).

RAGs bind symmetric bubble and heterologous loop DNA structures. Increasing concentrations of RAG complexes were incubated with [γ-³²P]ATP end-labeled 50-bp ds oligomers containing a symmetric bubble structure or a heterologous loop structure for 10 min at 37°C in buffer A in the presence 1 μM 45-bp double-stranded nonspecific DNA. The reaction products were then resolved on a 6% native polyacrylamide gel. In both the symmetric bubble structure and heterologous loop structure panels, the lanes are as follows: lanes 1 and 7, 50-bp DNA containing the non-B DNA structure treated with 0 ng RAGs; lanes 2 and 8, 10 ng RAGs; lanes 3 and 9, 25 ng RAGs; lanes 4 and 10, 50 ng RAGs; lanes 5 and 11, 100 ng RAGs; lanes 6 and 12, 200 ng RAGs; lane M, 50-bp ladder. The band resulting from the RAG binding with the specified structure is indicated by the arrowhead. The new band generated due to the nicked bubble structure is indicated by a small arrow near the bottom of the gel.

RAG binding occurs only when a non-B DNA structure or V(D)J recombination signal is present. RAGs (100 ng) were incubated with end-labeled 50-bp ds oligomer for 10 min at 37°C as described above. The reaction products were then resolved on a 6% native polyacrylamide gel. A. Comparison of RAG binding on duplex DNA, symmetric bubble, and 12-/23-signals. Single-stranded DNA was used in lanes 1 and 2. A 50-bp double-stranded DNA was prepared by annealing YM21 and SCR174 (lanes 3 and 4). A symmetric bubble structures (lanes 5 and 6) was prepared as described in Materials and Methods. The 12-signal was prepared by incubating KY28 and KY29, and the 23-signal was prepared by incubating KY36 and KY37. The shifted band is indicated by the arrow. Symm. Bubble, symmetric bubble. Core GST-RAGs were used for the RAG binding studies. Intervening lanes were removed between lanes 6 and 7. B. Comparison of RAG binding in the presence of GST and MBP-RAG preparations. “Cryptic signal” indicates that the sequence of the heptamer and nonamer varies from the standard V(D)J signal sequence (lanes 1 and 2). The core GST-RAGs were used in lanes 1 to 4. Core MBP-RAGs were used for binding studies in lane 5. The core MBP-RAG1 active site mutant and core MBP-RAG2 were used in lane 6. Intervening lanes of no interest were removed between lanes 4 and 5. C. The RAG binding mutant abolishes the binding ability of RAGs on symmetric bubble structures. The MBP-RAGs used are core double-MBP-RAGs (lane 3) and core double-MBP-RAG1 binding mutant/core double-MBP-RAG2 (lane 4). In all three panels, the shifted bands are indicated by arrows. The new band generated due to nicked bubble is indicated by a small arrow near the bottom of the gel.

P1 nuclease assay showing binding of RAGs to the single-stranded region of the symmetric bubble. Increasing concentrations of P1 nuclease were incubated with a [γ-³²P]ATP end-labeled 50-bp ds oligomers containing a symmetric bubble structure, with and without pretreatment with RAGs (100 ng of RAGs treated for 10 min at 37°C, in a volume of 15 μl) in buffer A for 15 min for 37°C. The reaction products were then resolved either on a 15% native polyacrylamide gel (A) or a 15% denaturing polyacrylamide gel (B). In both panels, lanes 1 to 5 contain no RAGs and lanes 6 to 10 are with RAGs. The shifted band due to the nicked bubble is indicated by an arrow in panel A. Lane M, 50-bp ladder; lane M′, 22-nt oligomer digested with Klenow to generate a ladder; lane O, [γ-³²P]ATP-labeled 22-nt oligomer. The bubble region is indicated by the bracket.