T the exact same time in individual cells. This tight regulation of V(D)J recombination could give a mechanism for preventing inter-locus rearrangement and for preventing the introduction of various DNA DSBs Catalase Autophagy inside the identical cell, which could otherwise constitute a threat to genome stability. Differences in RAG2-S365A Cleavage Do not Arise from Differences in Expression, Cleavage Efficiency, Recombination, or even a Defect in Repair It can be conceivable that the boost in bi-allelic, bi-locus cleavage that we detect in the RAG2S365A cells could result from an elevated level of mutant RAG2 protein. To investigate this possibility, we performed a western blot to evaluate protein levels in cycling and noncycling (STI571 treated) cells expressing HA-tagged wild-type and mutant RAG2-S365A constructs. As an more handle, we also analyzed cells expressing mutant RAG2T490A. The threonine 490 (T490) residue, Vessel Inhibitors Related Products located inside the C-terminal area of RAG2, is phosphorylated by Cdk2 upon entry in to the S phase from the cell cycle, causing RAG2 protein degradation (Li et al., 1996; Lin and Desiderio, 1993; Zhang et al., 2011). This phosphorylation event negatively regulates recombinase activity across the cell cycle, preventing RAG-mediated cleavage outside of G1. Working with antibodies against the HA tags, we could only detect the non-degradable mutant RAG2-T490A protein inside the untreated cycling cells (Figure 3A). In contrast, all 3 constructs gave rise to equivalent levels of RAG2 protein in the STI571-treated cells. We also analyzed expression by immunofluorescence in individual cells. The tagged proteins reveal equivalent enrichment of RAG2 in euchromatic regions of the nucleus in cells expressing wild-type versus mutant RAG2-S365A constructs (Figure 3B). Furthermore, cleavage efficiency on the mutant RAG2-S365A protein was comparable to wild-type RAG2, as judged by use of a pMX-INV-integrated substrate that generates GFP as a readout for recombination (Liang et al., 2002) in Rag2-/- v-Abl-transformed cells (Figure 3C). Consistent with these findings, we also detected comparable levels of Igk recombination in cells expressing wild-type and mutant RAG2-S365A by qPCR using a Jk1 primer in addition to a degenerate Vk primer (Figure 3D). Comparable outcomes had been obtained with semiquantitative PCR working with a Vk to Jk5 primer in untreated and STI-treated cells. Here, we also analyzed recombination in cells expressing mutant T490A RAG2. Only low levels of Igk recombination have been detected in the untreated cycling cells expressing wild-type and mutant RAG2-S365A constructs, whereas recombination occurred at slightly larger levels in the cells expressing the non-degradable RAG2-T490A construct (Figure 3E). In contrast, soon after STI571 treatment, we could detect no differences in the degree of Igk recombination in cells expressing wildtype versus mutant S365A or T490A RAG2. It ought to be noted that despite the fact that cells expressing the non-degradable T490A mutation have an elevated degree of protein in untreated cycling cells, this will not bring about bi-allelic Igk breaks (Figure S3; Table S4). Collectively, these data indicate that deregulated bi-allelic, bi-locus cleavage discovered in cellsAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptCell Rep. Author manuscript; offered in PMC 2017 October 30.Hewitt et al.Pageexpressing S365A-RAG2 can’t be attributed to alterations in protein levels or recombination efficiency. ATM deficiency and an absence from the C terminus of RAG2 lead to an unstable postcle.