Accessibility of Nucleosomal DNA to V(D)J Cleavage Is Modulated by RSS Positioning and HMG1  Jongbum Kwon, Anthony N Imbalzano, Adam Matthews, Marjorie A Oettinger  Molecular Cell  Volume 2, Issue 6, Pages 829-839 (December 1998) DOI: 10.1016/S1097-2765(00)80297-X

Figure 1 Experimental Design (A) DNA templates for nucleosome assembly. Nucleosomal templates for generating different rotational (left) and translational positions (right) of the 12- or 23RSS are represented to scale. Triangles represent the position of the RSS with the nonamer lying toward the point of the triangle. Predicted sizes of cleavage products visualized by 32P labeling at the 5′ end (*) are indicated. While the templates with a 12RSS are labeled at the 5′ end of the top strand, the templates with the 23RSS are labeled at both 5′ ends, allowing detection of the bottom strand of the signal ended fragment as well (designated as DSB). The expected position of the dyad is indicated. (B) Scheme used for analysis of nucleosome assembly and cleavage. See text for detailed description. Free DNA is used to refer to DNA that has not become incorporated into nucleosomes, while naked DNA denotes substrates never subjected to a nucleosome assembly protocol. Molecular Cell 1998 2, 829-839DOI: (10.1016/S1097-2765(00)80297-X)

Figure 2 Rotational Positioning of the 12 Signal for In Vitro–Assembled Nucleosomes (A) DNase I footprinting. DNase I digestion patterns for the top strand of nucleosomes (N), N12RSS+0, N12RSS+3, and N12RSS+6, are shown with those of corresponding naked DNA (D) and sequencing marker (G+A). The positions of the heptamer (hep) and nonamer (non) are indicated. (B) Schematic representation of rotational positioning of the 12-signal on the surface of the histone octamer. By comparing the 10 bp DNase I ladder with the sequencing marker, the relative orientation of the signal sequence was determined for each of the three nucleosomes; DNase I cleavage sites were aligned at the position of the double helix furthest from the surface of the histone octamer. Molecular Cell 1998 2, 829-839DOI: (10.1016/S1097-2765(00)80297-X)

Figure 3 V(D)J Cleavage Is Inhibited on Nucleosomal Templates (A) Analysis of the nucleosomal cleavage reaction by gel electrophoresis under native conditions. N12RSS+0 was incubated with buffer alone (−) or RAG1/2 (+). The position of the nucleosomes (NUC) and hairpin product (HP) are shown. (B) Denaturing gel analysis of the samples eluted from the gel in (A) (NUC, lanes 3 and 4) and from separate cleavage reactions of the naked 12RSS+0 template (DNA, lanes 1 and 2). The positions of the substrate, hairpin (HP), and nicked species are indicated along with schematic representations of the products. Filled circles indicate the position of 32P; triangles indicate the location of the RSS. Molecular Cell 1998 2, 829-839DOI: (10.1016/S1097-2765(00)80297-X)

Figure 4 HMG1 Stimulation of V(D)J Cleavage on Nucleosomal Templates Containing a 12RSS Is Sensitive to Rotational Position (A) Denaturing gel analysis. The indicated nucleosomes were incubated with no protein (lanes 2, 7, and 12), RAG1/2 alone (lanes 3, 8, and 13), or RAG1/2 plus two concentraions of HMG1 (0.2 μg/ml for lanes 4, 9, and 14; 1.0 μg/ml for lanes 5, 10, and 15). The cleavage of naked DNA of the corresponding nucleosome is shown in the first lane of each subset (D) (lanes 1, 9, and 17). (B) Representative native gel analysis. Reactions with 2.5 μg/ml of HMG1; lanes 3, 6, and 9 for each nucleosome are shown along with reactions containing no HMG1 and no RAG1/2 (lanes 1, 4, and 7) or RAG1/2 alone (lanes 2, 5, and 8). (C) HMG1 stimulates the cleavage of naked DNA equivalently for all three templates. Naked DNA of 12RSS+0, 12RSS+3, or 12RSS+6 was incubated with RAG1/2 alone (lanes 1, 4, and 7) or RAG1/2 plus 0.25 μg/ml (lanes 2, 5, and 8) or 2.5 μg/ml (lanes 3, 6, and 9) of HMG1 and analyzed on a denaturing gel. All labeling is as in Figure 3. Molecular Cell 1998 2, 829-839DOI: (10.1016/S1097-2765(00)80297-X)

Figure 5 HMG1 Stimulates Cleavage of 23RSS Nucleosomes (A) DNase I footprinting for N23RSS+0. DNase I digestion patterns for both top and bottom strands are shown. (B) Denaturing gel analysis of V(D)J cleavage. Indicated nucleosomes were incubated with no protein (lanes 7, 12, and 17), RAG1/2 alone (lanes 8, 13, and 18), or RAG1/2 plus 0.25 μg/ml (lanes 9, 14, and 19), 1.0 μg/ml (lanes 10, 15, and 20), or 2.5 μg/ml (lanes 11, 16, and 21) of HMG1. The cleavage for the corresponding naked DNA templates is shown in lanes 1–6. Because DNA was labeled at the 5′ ends of both strands, the signal-ended fragment (DSB) was also detectable in addition to the nick and hairpin after cleavage of naked DNA. Only the DSB product was recovered from the nucleosomal templates (see text for discussion). Molecular Cell 1998 2, 829-839DOI: (10.1016/S1097-2765(00)80297-X)

Figure 6 HMG1 Stimulates Cleavage of a 12RSS Equivalently when the RSS Is Positioned on the Dyad or Closer to the Entry/Exit Points of the Nucleosome The indicated nucleosomes were incubated with no protein (lanes 3, 10, and 17), RAG1/2 alone (lanes 4, 11, and 18), or RAG1/2 plus 0.25 μg/ml (lanes 5, 12, and 19), 1.0 μg/ml (lanes 6, 13, and 20), or 5.0 μg/ml (lanes 7, 14, and 21) of HMG1. V(D)J cleavage of the corresponding naked DNA templates is also shown (lanes 1, 2, 8, 9, 15, and 16). A schematic representation of nucleosomes N12RSS/TP1, N12RSS/TP2, and N12RSS/TP3 is shown below. The expected position of the single strong translational position of the histone core is represented by an oval. The 152 bp DNA template (horizontal line) containing a 12RSS (triangle) is schematically represented for each nucleosome with the position of the dyad indicated by an arrow. Other labeling is as in Figure 3. Molecular Cell 1998 2, 829-839DOI: (10.1016/S1097-2765(00)80297-X)

Figure 7 The Stimulation of V(D)J Cleavage by HMG1 of 23RSS Nucleosomal Templates Is Sensitive to the Position of the RSS Relative to the Dyad Indicated nucleosomes were incubated with no protein (lanes 3 and 10), RAG1/2 alone (lanes 4 and 11), or RAG1/2 plus 0.25 μg/ml (lanes 5 and 12), 1.0 μg/ml (lanes 6 and 13), or 5.0 μg/ml (lanes 7 and 14) of HMG1. The cleavage of the corresponding naked DNA is shown in lanes 1, 2, 8, and 9. DNA was labeled at both 5′ ends as in Figure 5. A schematic representation of nucleosomes N23RSS/TP1 and N23RSS/TP2 is also shown. The translational position of each nucleosome was determined as described in the text. The 23-signal is positioned over the dyad (N23RSS/TP1) or positioned with the nonamer end located 63 bp away from the dyad (N23RSS/TP2). Molecular Cell 1998 2, 829-839DOI: (10.1016/S1097-2765(00)80297-X)