Volume 34, Issue 1, Pages 13-25 (April 2009) Roles for NBS1 in Alternative Nonhomologous End-Joining of V(D)J Recombination Intermediates  Ludovic Deriano, Travis H. Stracker, Annalee Baker, John H.J. Petrini, David B. Roth  Molecular Cell  Volume 34, Issue 1, Pages 13-25 (April 2009) DOI: 10.1016/j.molcel.2009.03.009 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 NBS1 Is Required for RAG2FS361-Mediated Bypass of Coding Joint Deficiency in Prkdcscid/scid and Artemis−/− Fibroblasts (A and B) Representation of data from Table S1. SV40-immortalized mouse fibroblasts were transfected with a coding joint reporter substrate (pJH290) and the indicated RAG expression vectors. Plasmid DNA was harvested and used to transform bacteria; colonies were counted and used to calculate recombination frequencies. Results are averaged from three independent experiments. Error bars, SEM; p values were calculated using Student's two-tailed t test. Molecular Cell 2009 34, 13-25DOI: (10.1016/j.molcel.2009.03.009) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 TCR D2-J1 Coding Joint Formation in Artemis−/− Thymocytes Depends on NBS1 (A and B) Mouse thymus DNA (200 ng unless otherwise noted) analyzed by semiquantitative PCR for TCRδ D2-J1 rearrangements (R). Expected sizes of PCR products: 160 bp (coding joint), 1 kb (germline), and 304 bp (signal joint). (C) PCR products were quantified, and D2-J1 rearrangements were measured (as “D™2-J™1 coding joint/[D™2-J™1 coding joint + D™2-J™1 germline]”) and expressed as a percentage of recombination in wild-type samples. Results are averaged from n mice as indicated. Error bars, SEM; p values were calculated using Student's two-tailed t test. (D) D2-J1 signal joints in the same samples as in (B). Molecular Cell 2009 34, 13-25DOI: (10.1016/j.molcel.2009.03.009) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 NBS1 Is Required for RAG2FS361-Mediated Alternative Coding Joint Formation in NHEJ-Proficient Cells (A) Substrate designed to measure alternative NHEJ. Only a specific junction (deleting 10 nt from each end and using a 9 nt microhomology) allows expression of GFP. (B and C) SV40-immortalized mouse fibroblast cell lines were transfected with the indicated RAG expression vectors, and the alternative NHEJ reporter and GFP-positive cells were assayed by flow cytometry 60 hr later. Results are averaged from > three independent experiments. Error bars, SEM; p values were calculated using Student's two-tailed t test. Molecular Cell 2009 34, 13-25DOI: (10.1016/j.molcel.2009.03.009) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Effect of NBS1 on Signal Joint Formation in Prkdcscid/scid and Artemis−/− Fibroblasts (A and B) Representation of data from Table S2. SV40-immortalized mouse fibroblasts were transfected with a signal joint reporter substrate (pJH289) and the indicated RAG expression vectors. Plasmid DNA was harvested and used to transform bacteria; colonies were counted and used to calculate recombination frequencies. Results are averaged from at least three independent experiments. Error bars, SEM; p values were calculated using Student's two-tailed t test. Molecular Cell 2009 34, 13-25DOI: (10.1016/j.molcel.2009.03.009) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Cells Bearing Hypomorphic NBS1 Protein Exhibit Defects in Inversional V(D)J Recombination and Increased Hybrid Joint Formation (A) (Top panel) Schematic of pMX-INV substrate (top); coding end (CE) and signal end (SE); DSB intermediates; and signal, coding, and hybrid joint products (Bredemeyer et al., 2006). The long terminal repeats (LTR); packaging sequence (ψ); GFP cDNA; IRES-hCD4 cDNA (I-hCD4); 5′ 12 recombination signal and 3′ 23 recombination signal (filled and open triangles, respectively); pA, pB, and pC oligonucleotides; EcoRI restriction site; and C4 probe (black line) are indicated (adapted from Bredemeyer et al., 2006). (Bottom panel) Diagram of inversional and deletional (hybrid joint) recombination. (B) SV40-immortalized mouse fibroblasts with integrated pMX-INV were assayed by flow cytometry 60 hr after transfection with the indicated RAG expression vectors. The percentage of GFP-expressing cells is indicated. Results are averaged from three independent experiments ± SEM. (C) PCR analysis of CJ and HJ formation in the same experimental setting as in (B). Cells were harvested 60 hr posttransfection, and 200 ng or 20 ng of genomic DNA were used for PCR. PCR for IL-2 was used as control for template DNA concentration. (D) SV40-immortalized mouse fibroblasts of the indicated genotype were transfected with inversional substrate (pJH299) (see Figure S8A). At 48 hr after transfection, plasmid DNA was recovered and transformed into bacteria. Individual recombined plasmids from chloramphenicol-resistant colonies were PCR amplified to determine the proportion of inversional and hybrid joints. n = number of colonies analyzed; p values were calculated using a Fisher's exact test. Molecular Cell 2009 34, 13-25DOI: (10.1016/j.molcel.2009.03.009) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Lymphocytes Bearing Hypomorphic NBS1 Protein Exhibit Defects in Inversional V(D)J Recombination and Increased Hybrid Joint Formation (A) MACS-purified DN thymocytes from adult wild-type (+/+), Nbs1ΔB/+, or Nbs1ΔB/ΔB mice and total E17.5 embryonic thymocytes were infected with pseudotyped PMX-INV-GFP reporter viruses. After 48 hr, cells were stained with PE-anti-hCD4 and assayed by flow cytometry. Percentages shown are out of live thymocytes. The RAG-INV index was defined as the percentage of GFP+ cells divided by the percentage of total hCD4+ cells. (B) Results are averaged from n animals (either DN thymocytes from adult mice or total thymocytes from E17.5 embryos). Error bars, SEM; p values were calculated using Student's two-tailed t test. (C) PCR analysis of coding joint and hybrid joint formation in DN thymocytes from indicated adult mice. Cells were harvested 48 hr postinfection, and 400 ng or 100 ng of genomic DNA from each sample were PCR amplified. PCR for IL-2 was used as control for template DNA concentration. (D) Schematic showing the relative orientation of the Vκ6-23 and Jκ1 gene segments. RSS are shown as open triangles; arrows denote PCR primers. (E) PCR analysis of Vκ6-23 to Jκ1 hybrid joints in splenocytes of indicated mouse genotypes using 500 ng or 100 ng of genomic DNA. Molecular Cell 2009 34, 13-25DOI: (10.1016/j.molcel.2009.03.009) Copyright © 2009 Elsevier Inc. Terms and Conditions