RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre–B cell receptor (pre–BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre–BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre–B cells. Here, we show that RAG DSBs inhibit pre–BCR signals through the ATM- and NF-κB2–dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre–BCR signaling. This regulatory circuit prevents the pre–BCR from inducing additional Igl chain gene rearrangements and driving pre–B cells with RAG DSBs into cycle. We propose that pre–B cells toggle between pre–BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes

ATM Limits Incorrect End Utilization during Non-Homologous End Joining of Multiple Chromosome Breaks

Chromosome rearrangements can form when incorrect ends are matched during end joining (EJ) repair of multiple chromosomal double-strand breaks (DSBs). We tested whether the ATM kinase limits chromosome rearrangements via suppressing incorrect end utilization during EJ repair of multiple DSBs. For this, we developed a system for monitoring EJ of two tandem DSBs that can be repaired using correct ends (Proximal-EJ) or incorrect ends (Distal-EJ, which causes loss of the DNA between the DSBs). In this system, two DSBs are induced in a chromosomal reporter by the meganuclease I-SceI. These DSBs are processed into non-cohesive ends by the exonuclease Trex2, which leads to the formation of I-SceI–resistant EJ products during both Proximal-EJ and Distal-EJ. Using this method, we find that genetic or chemical disruption of ATM causes a substantial increase in Distal-EJ, but not Proximal-EJ. We also find that the increase in Distal-EJ caused by ATM disruption is dependent on classical non-homologous end joining (c-NHEJ) factors, specifically DNA-PKcs, Xrcc4, and XLF. We present evidence that Nbs1-deficiency also causes elevated Distal-EJ, but not Proximal-EJ, to a similar degree as ATM-deficiency. In addition, to evaluate the roles of these factors on end processing, we examined Distal-EJ repair junctions. We found that ATM and Xrcc4 limit the length of deletions, whereas Nbs1 and DNA-PKcs promote short deletions. Thus, the regulation of end processing appears distinct from that of end utilization. In summary, we suggest that ATM is important to limit incorrect end utilization during c-NHEJ

Pharmacological augmentation strategies for schizophrenia patients with insufficient response to clozapine : a quantitative literature review

Background. When schizophrenia patients have insufficient response to clozapine, pharmacological augmentation is often applied. This meta-analysis summarizes available evidence on efficacy of pharmacological augmentation of clozapine treatment in schizophrenia spectrum disorder. Methods. Only double-blind randomized controlled studies were included. Primary outcome measure was total symptom severity, and secondary outcome measures were subscores for positive and negative symptoms. Effect sizes were calculated from individual studies and combined to standardized mean differences (Hedges's g). Results. Twenty-nine studies reporting on 15 different augmentations were included. Significant better efficacy than placebo on total symptom severity was observed for lamotrigine, citalopram, sulpiride, and CX516 (a glutamatergic agonist). The positive effect of lamotrigine disappeared after outlier removal. The other positive findings were based on single studies. Significantly better efficacy on positive symptom severity was observed for topiramate and sulpiride. The effect of topiramate disappeared after outlier removal. Results for sulpiride were based on a single randomized controlled trial. Citalopram, sulpiride, and CX516 showed better efficacy for negative symptoms than placebo, all based on single studies. Conclusions. Evidence for efficacy of clozapine augmentation is currently scarce. Efficacy of lamotrigine and topiramate were both dependent on single studies with deviating findings. The effect of citalopram, sulpiride, and CX516 were based on single studies. Thus, despite their popularity, pharmacological augmentations of clozapine are not (yet) demonstrated to be superior to placebo