The ATM Kinase Restrains Joining of Both VDJ Signal and Coding Ends

Abstract

The evidence that ATM affects resolution of RAG-induced DNA double-strand breaks is profuse and unequivocal; moreover, it is clear that the RAG complex itself cooperates (in an undetermined way) with ATM to facilitate repair of these double-strand breaks by the classical nonhomologous end-joining pathway. The mechanistic basis for the cooperation between ATM and the RAG complex has not been defined, although proposed models invoke ATM and RAG2's C terminus in maintaining the RAG postcleavage complex. In this study, we show that ATM reduces the rate of both coding and signal joining in a robust episomal assay; we suggest that this is the result of increased stability of the postcleavage complex. ATM's ability to inhibit VDJ joining requires its enzymatic activity. The noncore C termini of both RAG1 and RAG2 are also required for ATM's capacity to limit signal (but not coding) joining. Moreover, potential phosphorylation targets within the C terminus of RAG2 are also required for ATM's capacity to limit signal joining. These data suggest a model whereby the RAG signal end complex is stabilized by phosphorylation of RAG2 by ATM.

Fig. 1. ATM inhibits both VDJ coding and signal end joining, but not joining of I-Sce1 induced DSBs

A. Diagram of fluorescent substrates utilized to detect signal joints, coding joints, end joining of I-Sce1 induced DSBs, or homologous recombination of I-Sce1 induced DSBs. B. Immunoblot of 293T cells transfected with GFP-tagged trR1 and wtR2 with either GFP-tagged ATM or DNA-PKcs, or vector only. C. Wild type 293T cells were transfected with the coding, signal, or I-Sce1 end joining substrates and RAG1, RAG2, or I-Sce1 as well as control plasmids (vect-kd, vect-ATM, Ndel-kd, Ndel-ATM) or expression plasmids encoding wild type ATM (wt ATM) or kinase inactive ATM (kd ATM). Cells were analyzed by flow cytometry; % of cells expressing CFP/RFP is indicated as % recombination. Results were compiled from four experiments, with each experiment performed in triplicate for each variable. *** Indicates P<.0001 in two-tailed unpaired t-tests. D. Immunoblot of wild type 293T cells transfected with wild type or kinase inactive ATM. E. 293T cells were transfected with the coding, signal, or I-Sce1 end joining or homologous recombination substrates and RAG1, RAG2, or I-Sce1 as well as control plasmid, wild type DNA-PKcs, or wild type ATM. Cells were analyzed by flow cytometry and analyzed as above. Results were compiled from four experiments. *** Indicates P<.0001. F. 293T cells were transfected with the signal joint substrate and RAG1, RAG2, and increasing concentrations of wild type ATM. Cells were analyzed by flow cytometry as in 1B. Results presented were compiled from three experiments.

Fig. 2. ATM limits accessibility of VDJ intermediates to other DNA modifying enzymes

A. PCR for signal joints of substrate plasmids recovered by alkaline lysis of ATM deficient 293T transfected with or without RAG1/2, with either kinase inactive ATM or wild type ATM, and Trex2 or control plasmid. Amplified products were digested or not with ApaLI prior to electrophoresis. Arrows depict position of cut and uncut signal joints. This experiment is representative of five independent experiments. * Indicates a non-specific amplification product. B. Individual signal joints from indicated transfections were cloned and tested for sensitivity to ApaLI. Signal joints that were resistant to ApaLI were sequenced.

Fig. 3. “Non-core” regions of both RAG1 and RAG2 are required for maximal ATM inhibition of VDJ joining

A. Diagram of RAG 1 and RAG2. B. 293T cells were transfected with the coding or signal end joining substrates and full length or core RAG1 and RAG2. For each RAG1/2 combination, assays were performed including kinase inactive ATM (open bars) or wild type ATM (hatched bars). Cells were analyzed as in Fig 1B. Results were compiled from nine experiments. *** Indicates P<.0001. C. 293T cells were transfected with the coding or signal end joining substrates and full length or core RAG2, and either full length RAG1, N-terminal deleted RAG1 (ndelR1), or C-terminal deleted RAG1 (cdelR1). Assays were performed including kinase inactive ATM (open bars) or wild type ATM (hatched bars). Cells were analyzed as in Fig 1B. Results were compiled from seven experiments. *** Indicates P<.0001 and * indicates P<.01. D. Immunoblot analyses of 293T cells transfected with wild type (WT), core (tr), C terminal deleted (CD) or N terminal deleted (ND) RAG1 or RAG2 as indicated. E. PCR for signal joints of substrate plasmids recovered by alkaline lysis of ATM deficient 293T transfected with indicated plasmids. Amplified products were digested or not with ApaLI prior to electrophoresis. Arrows depict position of cut and uncut signal joints. This experiment is representative of three independent experiments. * Indicates a non-specific amplification product.

Fig. 4. Ablating potential phosphorylation sites in RAG2’s C-terminus attenuates inhibition of signal end joining by ATM

A. Diagram of RAG2. Comparison of 118 RAG2 protein sequences revealed many highly conserved potential DNA-PK/ATM phosphorylation targets (denoted with red lines for S-T/hyd and S-T/Q, or blue lines for S-T/P, and listed below). For each cluster (I-VI) all sites in each cluster were substituted with alanine (i.e. 1 S>A) or aspartic acid (i.e. 1 S>D). (+) Denotes sites previously shown to be phosphorylated (61). B. 293T cells were transfected with the coding or signal end joining substrates and full length RAG1 or C-terminal deleted RAG1 (cdelR1) in conjunction with full length RAG2, 4X, 5X, 11X, 12X, or core RAG2. Results were compiled from seven experiments. *** Indicates P<.0001 and * indicates P<.01. Pairwise statistical analyses was also performed comparing cdelR1/wt with cdelR1 and each of the phosphorylation mutants for both coding and signal joining. The differences for all except 4XSA signal joining, were highly statistically significant, P<.0001. C. Immunoblot showing expression of RAG. D. PCR for signal joints of substrate plasmids recovered by alkaline lysis of ATM deficient 293T transfected with indicated plasmids. Amplified products were digested or not with ApaLI prior to electrophoresis. Arrows depict position of cut and uncut signal joints. This experiment is representative of three independent experiments. * Indicates a non-specific amplification product.

Fig. 5. Blocking potential ATM phosphorylation sites in RAG2 restores episomal signal joining in DNA-PKcs−/− 293T cells

Left. DNA-PKcs deficient 293T cells were transfected with the signal end joining substrates and full length RAG1 or C-terminal deleted RAG1 (cdelR1) in conjunction with full length RAG2, 4X, 5X, 11X, 12X, or core RAG2. Results were compiled from seven experiments. Right. Immunoblot of DNA-PKcs deficient 293T cells transfected with the indicated plasmids.