V(D)J recombination and class switch recombination employ overlapping but distinct non-homologous end-joining (NHEJ) pathways to repair DNA double strand break (DSB) intermediates. 53BP1 is a DNA damage response protein that is rapidly recruited to sites of chromosomal DSBs, where it appears to function in a subset of ataxia-telangiectasia mutated (ATM) kinase, H2AX- and MDC1- dependent events1,2. A 53BP1 dependent end joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination CSR3, 4. Here, we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1 deficient lymphocytes distinct from that found in classical NHEJ-, H2AX-, MDC1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of un-repaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes bearing antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long range joining of DNA breaks.

DNA double strand breaks are generated by genotoxic agents and by cellular endonucleases as intermediates of several important physiologic processes. The cellular response to genotoxic DNA breaks includes the activation of transcriptional programs known primarily to regulate cell cycle checkpoints and cell survival1–5. DNA double strand breaks are generated in all developing lymphocytes during the assembly of antigen receptor genes, a process that is essential for normal lymphocyte development. Here we demonstrate that these physiologic DNA breaks activate a broad transcriptional program. This program transcends the canonical DNA double strand break response and includes many genes that regulate diverse cellular processes important for lymphocyte development. Moreover, the expression of several of these genes is regulated similarly in response to genotoxic DNA damage. Thus, physiologic DNA double strand breaks provide cues that can regulate cell-type-specific processes not directly involved in maintaining the integrity of the genome, and genotoxic DNA breaks could disrupt normal cellular functions by corrupting these processes.

The diagnosis of COVID‐19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS‐CoV‐2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single‐center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR‐positive SARS‐CoV‐2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%‐71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR‐positive patients with a total of 197 specimens tested by enzyme‐linked immunosorbent assay for IgM, IgG, and IgA anti‐SARS‐CoV‐2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time‐dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Lymphocyte antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG endonuclease introduces DNA DSBs at two recombining gene segments to form broken DNA coding end pairs and signal end pairs. These paired DNA ends are joined by proteins of the nonhomologous end-joining (NHEJ) pathway of DSB repair to form a coding joint and signal joint, respectively. RAG DSBs are generated in G1-phase developing lymphocytes, where they activate the ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases to orchestrate diverse cellular DNA damage responses including DSB repair. Paradoxically, although Atm and DNA-PKcs both function during coding joint formation, Atm appears to be dispensible for signal joint formation; and although some studies have revealed an activity for DNA-PKcs during signal joint formation, others have not. Here we show that Atm and DNA-PKcs have overlapping catalytic activities that are required for chromosomal signal joint formation and for preventing the aberrant resolution of signal ends as potentially oncogenic chromosomal translocations.

Canonical chromosomal translocations juxtaposing antigen receptor genes and oncogenes are a hallmark of many lymphoid malignancies. These translocations frequently form through the joining of DNA ends from double-strand breaks (DSBs) generated by the recombinase activating gene (RAG)-1 and -2 proteins at lymphocyte antigen receptor loci and breakpoint targets near oncogenes. Our understanding of chromosomal breakpoint target selection comes primarily from the analyses of these lesions, which are selected based on their transforming properties. RAG DSBs are rarely resolved aberrantly in wild-type developing lymphocytes. However, in ataxia telangiectasia mutated (ATM)-deficient lymphocytes, RAG breaks are frequently joined aberrantly, forming chromosomal lesions such as translocations that predispose (ATM)-deficient mice and humans to the development of lymphoid malignancies. Here, an approach that minimizes selection biases is used to isolate a large cohort of breakpoint targets of aberrantly resolved RAG DSBs in Atm-deficient lymphocytes. Analyses of this cohort revealed that frequently, the breakpoint targets for aberrantly resolved RAG breaks are other DSBs. Moreover, these nonselected lesions exhibit a bias for using breakpoints in cis, forming small chromosomal deletions, rather than breakpoints in trans, forming chromosomal translocations.ataxia telangiectasia mutated ͉ chromosomal translocation ͉ DNA double-strand break repair ͉ V(D)J recombination D ouble-strand breaks (DSBs) in DNA are generated by genotoxic agents and cellular endonucleases as intermediates in several important physiologic processes including V(D)J recombination, Ig class switch recombination (CSR), DNA replication, gene transcription, and meiosis. DNA DSBs activate a highly conserved cellular response that prevents cell cycle progression, initiates repair of the broken DNA ends, and promotes apoptosis of cells with persistent un-repaired DSBs (1, 2). Broken DNA ends from a single DSB are usually rejoined; however, in some processes, such as V(D)J recombination and CSR, DNA ends arising from two DSBs are joined in a regulated fashion, generating a new gene product (1-4). Broken DNA ends from distinct DSBs can also be joined aberrantly, leading to the formation of potentially dangerous chromosomal lesions such as translocations, deletions, and inversions.All developing lymphocytes generate programmed DNA DSBs during V(D)J recombination, a process that joins variable (V), joining (J), and in some cases, diversity (D) gene segments to generate the second exon of antigen receptor genes (4). The V(D)J recombination reaction is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which together form the RAG endonuclease (5). RAG introduces DNA DSBs at the border of two recombining gene segments and their flanking RAG recognition sequences, termed recombination signals (RSs) (5). DNA cleavage by RAG occurs after an appropriate RS pair (12/23 compatible) forms a synaptic complex, generating

Introduction: The diagnosis of COVID-19 requires integration of clinical and laboratory data. SARS-CoV-2 diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. Methods: A single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. Results: Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70-71% from days 9-11, and 30% at day 21. In contrast, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after day 7, >80% after day 12, and 100% by day 21. Conclusion: PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Translocations involving the T cell receptor alpha/delta (TCRα/δ) chain locus, which bring oncogenes in the proximity of the TCRα enhancer, are one of the hallmark features of human T cell malignancies from ataxia telangiectasia (AT) and non-AT patients. These lesions are frequently generated by the fusion of DNA breaks at the TCRα/δ locus to a disperse region centromeric of the immunoglobulin heavy chain (IgH) locus. Aberrant VDJ joining accounts for TCRα/δ associated DNA cleavage, but the molecular mechanism that leads to generation of the “oncogene partner” DNA break is unclear. Here we show that in ATM deficient primary mouse T cells, IgH/TCRα/δ fusions arise at a remarkably similar frequency as in human AT lymphocytes. Recombinase-activating gene (RAG) is responsible for both TCRα/δ as well as IgH associated breaks on chromosome 12 (Chr12), which are subject to varying degrees of chromosomal degradation. We suggest a new model for how oncogenic translocations can arise from two non-concerted physiological DSBs.

Background: To seek insights into the pathogenesis of chronic active antibody-mediated rejection (CAMR), we performed mRNA analysis and correlated transcripts with pathologic component scores and graft outcomes. Methods: We utilized the NanoString nCounter platform and the Banff Human Organ Transplant gene panel to quantify transcripts on 326 archived renal allograft biopsy samples. This system allowed correlation of transcripts with Banff pathology scores from the same tissue block as well as correlation with long-term outcomes. Results: The only pathology score that correlated with AMR pathways in CAMR was peritubular capillaritis (ptc). C4d, cg, g, v, i, t, or ci scores did not correlate. DSA-negative CAMR had lower AMR pathway scores than DSA-positive CAMR. Transcript analysis in non-CAMR biopsies yielded evidence of increased risk of later CAMR. Among 108 patients without histologic CAMR, 23 developed overt biopsy-documented CAMR within 5 years and as a group had higher AMR pathway scores (p=3.4x10-5). Random forest analysis correlated three-year graft loss with elevated damage, innate immunity, and macrophage pathway scores in CAMR and TCMR.. Graft failure in CAMR was associated with TCMR transcripts but not with AMR transcripts, and graft failure in TCMR was associated with AMR transcripts but not with TCMR transcripts. Conclusions: Peritubular capillary inflammation and DSA are the primary drivers of AMR transcript elevation. Transcripts revealed subpathological evidence of AMR, which often preceded histological CAMR and subpathological evidence of TCMR that predicted graft loss in CAMR.