Roles for NBS1 in alternative nonhomologous end-joining of V(D)J recombination intermediates

Abstract

Recent work has highlighted the importance of alternative, error-prone mechanisms for joining DNA double-strand breaks (DSBs) in mammalian cells. These noncanonical, nonhomologous end-joining (NHEJ) pathways threaten genomic stability but remain poorly characterized. The RAG postcleavage complex normally prevents V(D)J recombination-associated DSBs from accessing alternative NHEJ. Because the MRE11/RAD50/NBS1 complex localizes to RAG-mediated DSBs and possesses DNA end tethering, processing, and joining activities, we asked whether it plays a role in the mechanism of alternative NHEJ or participates in regulating access of DSBs to alternative repair pathways. We find that NBS1 is required for alternative NHEJ of hairpin coding ends, suppresses alternative NHEJ of signal ends, and promotes proper resolution of inversional recombination intermediates. These data demonstrate that the MRE11 complex functions at two distinct levels, regulating repair pathway choice (likely through enhancing the stability of DNA end complexes) and participating in alternative NHEJ of coding ends.

Figure 1. NBS1 is required for RAG2^FS361-mediated bypass of coding joint deficiency in Prkdc^scid/scid and Artemis^−/− fibroblasts

(A, B) Supplementary Table 1 is graphically presented. 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 3 independent experiments; error bars, s.e.m.; P values were calculated using Student’s two-tailed t-test.

Figure 2. TCR Dδ2-Jδ1 coding joint formation in Artemis^−/− thymocytes depends on NBS1

(A–D) Mouse thymus DNA (200 ng unless otherwise noted) analyzed by semi-quantitative PCR for TCR Dδ2-Jδ1 rearrangements (R). Expected sizes of PCR products: 160 bp (coding joint), 1 kb (germline), 304 bp (signal joint). (C) PCR products were quantified and Dδ2-Jδ1 rearrangements were measured (as “Dδ2-Jδ1 coding joint/(Dδ2-Jδ1 coding joint + Dδ2-Jδ1 germline”) and expressed as a percentage of recombination in wild-type samples. Results are averaged from n mice as indicated; error bars, s.e.m.; P values were calculated using Student’s two-tailed t-test. (D) Dδ2-Jδ1 signal joints in the same samples as in (B)

Figure 3. NBS1 is required for RAG2^FS361-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, 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 h later. Results are averaged from >3 independent experiments; error bars, s.e.m.; P values were calculated using Student’s two-tailed t-test.

Figure 4. Effect of NBS1 on signal joint formation in Prkdc^scid/scid fibroblasts

Supplementary Table 2 is graphically presented. 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 6 independent experiments; error bars, s.e.m.; P values were calculated using Student’s two-tailed t-test.

Figure 5. Cells bearing hypomorphic NBS1 protein exhibit defects in inversional V(D)J recombination and increased hybrid joint formation

(A) 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), the pA, pB, and pC oligonucleotides, EcoRI restriction site and C4 probe (black line in top part) are indicated (Adapted from Bredemeyer et al. 2006) (upper panel). Lower panel, diagram of inversional and deletional (hybrid joint) recombination. (B) SV40-immortalized mouse fibroblasts with integrated pMX-INV were assayed by flow cytometry 60 h after transfection with the indicated RAG expression vectors. The percentage of GFP-expressing cells is indicated. Results are averaged from three independent experiments ± s.e.m. (C) PCR analysis of CJ and HJ formation in same experimental setting as in (B). Cells were harvested 60 hours post transfection, and 200 ng or 20ng 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 Supplementary Fig. 8A). 48 h 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.

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 (+/+), Nbs1^ΔB/+ or Nbs1^ΔB/ΔB mice and total E17.5 embryonic thymocytes were infected with pseudotyped PMX-INV-GFP reporter viruses. After 48 hours, cells were stained with PE-anti-hCD4 and assayed 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, s.e.m.; P values were calculated using Student’s two-tailed t-test. (C) PCR analysis of CJ and HJ formation in DN thymocytes from indicated adult mice. Cells were harvested 48 hours postinfection, and 400 ng or 100ng 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 shown as open triangles; arrows show denote PCR primers. (E) PCR analysis of Vκ6-23 to Jκ1 hybrid joints in splenocytes of indicated mouse genotypes using 500 ng or 100ng of genomic DNA.