A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

Coster, Gideon; Gold, Ayala; Chen, Darlene; Schatz, David G.; Goldberg, Michal

doi:10.1074/jbc.m112.402487

Cited by 23 publications

(25 citation statements)

References 68 publications

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“…Induction of RAG expression by these conditions is indicated as STI-571 + . Expression of RAG2 was evidenced by increased immunostaining with clone 39 rabbit monoclonal antibody specific to RAG2 (21) (Supplemental Figure 1A), and the appearance of a 60 kDa band in RAG2 immunoblots of whole cell lysates specific to samples grown in STI-571 (Supplemental Figure 1B). …”

Section: Methodsmentioning

confidence: 99%

Spatio-temporal regulation of RAG2 following genotoxic stress

et al. 2015

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V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The coexistence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex. Using cellular imaging and subcellular fractionation approaches, we show that formation of DSBs by treating cells with DNA damaging agents causes export of nuclear RAG2. Within the cytoplasm, RAG2 exhibited substantial enrichment at the centrosome. Further, RAG2 export was sensitive to inhibition of ATM, and was reversed following DNA repair. The core region of RAG2 was sufficient for export, but not centrosome targeting, and RAG2 export was blocked by mutation of Thr490. In summary, DNA damage triggers relocalization of RAG2 from the nucleus to centrosomes, suggesting a novel mechanism for modulating cellular responses to DSBs in developing lymphocytes.

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Section: Methodsmentioning

confidence: 99%

Spatio-temporal regulation of RAG2 following genotoxic stress

et al. 2015

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“…After three 3-min washes, proteins were eluted with 10 mM glutathione and SDS-PAGE sample buffer and analyzed by SDS-PAGE. Western blots were developed with anti-GST (Santa Cruz Biotechnology), anti-MBP (Cell Signaling), or anti-RAG2 monoclonal antibody (mAb) number 39 (26).…”

Section: Methodsmentioning

confidence: 99%

“…Samples in loading buffer (2% SDS) were heated to 95°C for 20 min, spun at 14,000 rpm for 5 min, fractionated by SDS-PAGE (8% acrylamide; 100 V, 1.5 h), and transferred to polyvinylidene difluoride membranes (Bio-Rad) by electroblotting (120 V, 0.5 h). Membranes were incubated with either anti-RAG2 mAb number 39 or anti-RAG1 mAb number 23 (26), diluted 1:500 in PBS overnight at 4°C, washed, and incubated with secondary antibody (goat anti-rabbit IgG (HϩL) alkaline phosphatase, Jackson ImmunoResearch) diluted 1:1,000 in PBS for 1 h. Signals were detected using ECF chemiluminescent detection substrate (GE Healthcare). Membranes were imaged by Pharos Fx Plus (Bio-Rad); bands were quantitated using Image Lab version 4.1 (Bio-Rad), and local background (determined from a region of membrane just above each band) was subtracted to yield the signal for each fluorescent band.…”

Section: Methodsmentioning

confidence: 99%

Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2

Zhang

Shetty

Surleac

et al. 2015

Journal of Biological Chemistry

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Background: The RAG1-RAG2 interaction is critical for V(D)J recombination but is poorly understood. Results: The RAG1-RAG2 interaction has a binding constant of ϳ0.4 M and requires only a small portion of RAG1. Conclusion: RAG1 and RAG2 interact with modest affinity using regions of RAG1 flanking the RAG1 catalytic region. Significance: Inefficient association of RAG1 with RAG2 could help limit damage to the genome.

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“…The antibodies and procedures used for the ChIP assay were described in detail previously (25,33). In brief, cells were harvested after 0, 3, 6, 24, and 48 h of STI-571 induction; cross-linked with 1% HCHO (formaldehyde) for 15 min at room temperature (RT); quenched with 0.125 M glycine; washed; and frozen as cell pellets.…”

Section: Coding Segmentmentioning

confidence: 99%

“…The pellets were then resuspended in radioimmunoprecipitation assay (RIPA) buffer containing 0.8 M NaCl and sonicated. The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody (33), anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore). After reversal of crosslinks and purification of the DNA (DNA Clean concentrator; Zymo), input and immunoprecipitated DNAs were quantitated by duplicate trials of TaqMan qPCR Qiagen HotStart Taq with a 3000 XP thermocycler (Stratagene).…”

Section: Coding Segmentmentioning

confidence: 99%

Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination

Shetty

Schatz

2015

Molecular and Cellular Biology

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b V(D)J recombination is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Here, we used RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for RAG recruitment to chromatin. Using a catalytic mutant form of RAG1 to prevent recombination, we did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous J gene segments over a 48-h time course. Using retroviral recombination substrates, we found that RAG1 was recruited inefficiently to substrates lacking an RSS or containing a single RSS, better to substrates with two 12-bp RSSs (12RSSs) or two 23-bp RSSs (23RSSs), and more efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding, and correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly re-creates the nonamer, was ineffective in recruiting RAG1. Our findings suggest that 12RSS-23RSS cooperation (the "12/23 rule") is important not only for regulating RAG-mediated DNA cleavage but also for the efficiency of RAG recruitment to chromatin. T he adaptive immune system relies on V(D)J recombination to assemble variable (V), diversity (D), and joining (J) antigen receptor gene segments in generating a diverse repertoire of immunoglobulins and T cell receptors. The process is initiated by the recombination-activating gene (RAG) recombinase, a protein complex consisting of the enzymes encoded by RAG1 and RAG2 (1, 2). RAG1 is 1,040 amino acids long, with a core region spanning amino acids 384 to 1008 that contains recombination signal sequence (RSS) and RAG2 binding activity as well as the catalytic residues responsible for DNA cleavage (3-7). RAG2 is 527 amino acids long, with a core region (amino acids 1 to 387) that interacts with and facilitates DNA binding and cleavage by RAG1 but which has no DNA binding activity on its own (3,4,8,9). The RAG2 noncore region contains a plant homeodomain (PHD) finger that plays an important role in chromatin binding through interactions with the histone H3 N-terminal tail when lysine 4 is trimethylated (H3K4me3) (10-12).The site of recombination is specified by the RSS that immediately flanks each V, D, and J gene segment (Fig. 1A). The RSS consists of a well-conserved 7-bp sequence called the heptamer (consensus sequence of 5=-CACAGTG-3=) and an AT-rich 9-bp sequence called the nonamer (consensus sequence of 5=-ACAAA AACC-3=) separated by a poorly conserved spacer whose length is either 12 or 23 bp (13-15) (Fig. 1B). An RSS with a spacer 12 bp long is defined as a 12RSS, while one with a spacer 23 bp long is defined as a 23RSS (13).The consensus RSS is the most efficiently recombined sequence and therefore has been used for most in vitro experiments; however, endogenous RSSs often deviate from the consensus at multiple hept...

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A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

Cited by 23 publications

References 68 publications

Spatio-temporal regulation of RAG2 following genotoxic stress

Spatio-temporal regulation of RAG2 following genotoxic stress

Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2

Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination

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