Thymocytes number (x10 6


ABLki: ATMki: ABLki: ATMki: 
+ + + + + + + + + - + - + - + - + - Ku80 -/-
- The reference sequence for 5' and 3' coding ends generated from the pMX-DEL CJ substrate is shown at the top. 
+ + -+ + -+ + -+ + -+ + -+ + -+ + -+ + --+ --+ --+ --+ -- + - -+ -- + --+ WT ( 
C µ C α C γ 1 V h D h J h C µ Dh 4-1 Jh 4 Jh 3 Jh 2 Jh 1 b C 14189LN 13111LN 14821LN 15510K 15510K 15510K 14189LN 14189LN 
When coding ends are resected beyond the 15 nucleoYdes shown in the reference sequence, the number of nucleoYdes resected is indicated in brackets along with the reference sequence flanking the resected end (in italics). N-and P-nucleoYde addiYons and microhomologies (MH) are indicated. * For sequences n° 7 and 35, the coding joints are located 205 and 166 bp downstream of RSS-12 respecYvely, potenYally corresponding to a miscleavage or a single cut at the RSS-23 followed by extensive resecYon. UR: unrearranged sequence. 

Supplementary Table 2a




  Normal lymphocyte development in Rag2 c/c Atm -/-animals. Lymphocyte development in WT, Rag2 c/c , Atm -/-, and Rag2 c/c Atm -/-mice. a. Total splenocytes (leS panel) and thymocytes (right panel) numbers. Each dot represents one mouse and bars indicate the median. WT, Rag2 c/c and Rag2 c/c Atm -/-mice are on mixed B6/129 backgrounds, whereas Atm -/-mice are on pure 129 background. b.c. Analysis of B cell development. b. RepresentaYve FACS analysis of bone marrow and spleen using B cell markers. Numbers on plots represent percentages of total cells. c. Percentage of bone marrow CD43 -B220 lo CD19 + IgM -pre-B cells (upper panel) and total number of splenic CD19 + IgM + B cells (bo]om panel). d.e. Analysis of T cell development. d. RepresentaYve FACS analysis of thymus and spleen using T cell markers. e. Total number of thymic DP cells (upper panel) and splenic CD3 + TCRβ + T cells (bo]om panel). Histograms represent means ± SD from at least 5 mice of each genotype. * 0.01 ≤ p < 0.05. 




  Supplementary Figure 2. Analysis of endogenousIgk rearrangements in v-abl pro-B cell lines. a. Southern blot with JKIII probe of EcoRI/SacI digested DNA from v-abl pro-B cell lines untreated and treated 72 or 96 hours with ABLki. b. Southern blot with JKIII probe of EcoRI/SacI digested DNA from v-abl pro-B cell lines treated for 72h with ABLki with or without ATMki. Arrows indicate normal rearrangements. Jk 1-5 CEs are indicated in red. 


Supplementary

  Figure 3. 53BP1 foci co-localize with γH2AX. a. RepresentaYve fluorescent light microscopy images of immuno-3D FISH analysis conducted on ABLkitreated Rag2 c/c XLF -/-v-abl pro-B cell lines. Nuclei were stained with 53BP1 (green) and γH2AX (pink). Images show nuclei with non coincident and coincident 53BP1/γH2AX signals. b. 




  foci co-localize with the Igk locus. a. RepresentaYve fluorescent light microscopy images of immuno-3D FISH analysis conducted on ABLkitreated on Rag2 c/c XLF -/-v-abl pro-B cell lines. Nuclei were stained with 53BP1 (green) and subsequently hybridized with the centromeric Igk BAC probe (Igk C, red). Images show nuclei with non-coincident and coincident 53BP1/Igk signals. b. Table indicates the number (%) of nuclei harboring non-coincident and coincident 53BP1/Igk signals among total nuclei of each cell class. assays in v-abl pro-B cell lines. a. DeterminaYon of opYmal dose of PD-180970 to induce V(D)J recombinaYon and preserve cell survival aSer 96h of treatment in WT v-abl pro-B cell lines carrying bcl-2 and pMX-INV transgenes (12096-MX/H) or not (12096). b. Pro-B cells of various genotypes were treated with PD-180970 (0.3µM) for 96h, with or without pre-incubaYon with KU55933 ATM inhibitor (ATMki, 15µM), and assayed for pMX-INV rearrangement by FACS analysis (numbers indicate percentage of GFP posiYve cells). 




  of pMX-INV rearrangements in v-abl pro-B cell lines by Southern bloWng. The indicated v-abl pro-B cell lines containing the pMX-INV substrate were treated for 72h with ABLki with or without ATMki and assayed by Southern blopng; EcoRV/NcoI digest -C4 probe (a) and EcoRV digest -pMX-INV rearrangement in Rag2 c/c Atm -/-v-abl pro-B cells. v-abl pro-B cell lines of indicated genotypes treated with ABLki for 0, 72h and 96h were assayed for PMX-INV rearrangement by flow cytometry, with the percentage of GFP expressing cells indicated. Histograms represent the means ± SD of 4 Atm -/-(160, 162, 13560, 13563), 6 Rag2 c/c (56, 145, 12018, 12019, 13570, 13572) and 3 Rag2 c/c Atm -/-(144, 13571, 13573) independent cell lines. analysis of pMX-DEL CJ coding joints in v-abl pro-B cell lines. a. SchemaYc of PCR approach for analyzing pMX-DEL CJ coding joints (CJs). The pC and IRES REV5 primers used to amplify CJs and the IR4 oligonucleoYde probe for Southern blot analysis are indicated. b. Southern blot with IR4 probe of pMX-DEL CJ PCR. WT and Rag2 c/c XLF -/-v-abl pro-B cell lines were treated for 72h with ABLki with or without ATMki. Serial five-fold diluYons of genomic DNA were amplified and run on a 1.2% agarose gel. Ethidium bromide gel (middle panel) has been transferred on a membrane and blo]ed with IR4 probe (top panel). Il-2 gene PCR was used as a loading control (bo]om panel). 




  Rag2 c/c (12018) XLF -/-(12004) Rag2 c/c XLF -/-(12009, 12013) of coding joint sequences in v-abl pro-B cell lines. Microhomology uYlizaYon (% of sequences with microhomologies ≥ 1bp, top panel; microhomology length distribuYon among the same sequences, bo]om panel) in pMX-DEL CJ CJs sequenced from indicated v-abl pro-B cell lines. 




  c Cernu -/-p53 -/- Control ** * p53 -/-, n=15, 20.3 weeks XLF -/-p53 -/-, n=15, 15 weeks Control Rag2 c/c XLF -/-p53 -/-, n=22, 11.55 weeks Rag2 c/c p53 -/-, n=28, 12.4 weeks Supplementary Figure 10. Analysis of Rag2 c/c XLF -/-p53 -/-mice survival. Kaplan-Meier survival curves of WT controls, p53 -/ -(n=15), XLF -/-p53 -/-(n=15), Rag2 c/c p53 -/-(n=28) and Rag2 c/c XLF -/-p53 -/-mice (n=22). * 0.01 ≤ p < 0.05, ** 0.001 ≤ p < 0.01. 




  (a) and Rag2 c/c XLF -/-:DEL CJ (b) v-abl pro-B cells. 


Rag -/- (129F) Cells with 1 53BP1 focus non coincident with γH2AX

  
	1 53BP1 focus					
	2 53BP1 foci					
		Non coincident	1 coincident	2 coincident
				741 (47.1%)	683 (28.1%)	374 (74.2%)	294 (77.4%)
		1 coincident with γH2AX	831 (52.9%)	1748 (71.9%)	130 (25.8%)	86 (22.6%)
			Total	1572	2431	504	380
		non coincident with γH2AX	35 (31.5%)	231 (19.0%)	121 (94.6%)	167 (100%)
	Cells with 2 53BP1 foci	1 coincident with γH2AX	41 (36.9%)	433 (35.7%)	7 (5.4%)	0
		2 coincident with γH2AX	35 (31.5%)	549 (45.2%)	0	0
			Total	111	1213	128	167

Cell class Coincidence WT (12095) Rag2 c/c XLF -/- (12009) Rag2 c/c XLF -/- (12013) Rag -/- (129M) Nuclei with 1 53BP1 focus non coincident with Igk 389

  
		(38.4%)	318 (20.1%)	291 (19.4%)	172 (68.3%)
	1 coincident with Igk	623 (61.6%)	1266 (79.9%)	1210 (80.6%)	80 (31.7%)
	Total	1012	1584	1501	252

Table indicates the number (%) of nuclei harboring non coincident and coincident 53BP1/γH2AX signals among total nuclei of each cell class.b 
Nuclei with 2 53BP1 foci non coincident with Igk 32

  
	(22.9%)	29 (3.1%)	61 (6.6%)	30 (39.0%)

1 coincident with Igk 83

  
	(59.3%)	255 (26.8%)	316 (34.1%)	47 (61.0%)

2 coincident with Igk 25

  
		(17.9%)	666 (70.1%)	551 (59.4%)	0
	Total	140	950	928	77

Table 2 . Coding joint sequences from WT:DEL CJ

 2 Table indicates the mean and SD of the experiments detailed in table a. 


		t(15;12)	t(6;15)	t(6;15)
		Pvt1/Igh	IgkV/Nsmce2	IgkJ1/Nsmce2
		-	-	14189LN	-
	Breakpoint	Breakpoint		Breakpoint

StaTsTcal analysis (Fisher exact test) Supplementary Table 3b Supplementary Table 3. Genomic instability at the Igk locus in

  
	Supplementary Table 2b			
	Seq Nb				5'CE		MH		P/N	3'CE
					TAATAGGAACGTCTG			CAGCCTACAATTCCG
	7* Genotype WT Supplementary Table 3a TCTGGTGTCAAA TCTGGTGT CTGGTGTCAAAAA CTGGTGTC GTGTCAAAAA GTGTCAAA GTCCGCCGACAC GTCCGCCG Cell line # Genotype Cell line # Genotype Experiment Expriment 1 (1) 12095 WT Expriment 2 (2) 12096 (1) TOTAL 165 WT 12096 (2) Expriment 1 TOTAL Rag2 c/c (1) 165 TOTAL Expriment 2 Rag2 c/c Expriment 3 12018 (1) TOTAL (2) 12019 Expriment 1 TOTAL Rag2 c/c 12018 (2) 13572 Expriment 2 XLF -/-Expriment 3 XLF -/-TOTAL TOTAL 12004 Expriment 1 TOTAL XLF -/-12005 Expriment 2 Rag2 c/c XLF -/-TOTAL Rag2 c/c XLF -/-Expriment 3 12013 Expriment 4 TOTAL 12009 TOTAL Rag2 c/c XLF -/-14837 Expriment 1 p53 -/-6920.1 6943.2 TOTAL 6920.1 Expriment 2 p53 -/-Expriment 3 TOTAL 6943.2 TOTAL p53 -/-15307 Expriment 1 Rag2 c/c p53 -/-4566.7 XLF -/-p53 -/-TOTAL Expriment 2 Rag2 c/c p53 -/-4566.7 Rag2 c/c p53 -/-Expriment 3 15259 Expriment 4 TOTAL XLF -/-p53 -/-15293 TOTAL Rag2 c/c XLF -/-p53 -/-TOTAL Expriment 1 TOTAL Rag2 c/c XLF -/-p53 -/-14838 Expriment 2 14840 XLF -/-p53 -/-Expriment 3 Rag -/-p53 -/-6527.2 TOTAL Expriment 4 Rag -/-p53 -/-6527.2 TOTAL	TAATAGGAA TAATAGGAACGT TAATAGGAACGT TAATAGGAACG TAATAGGAACG TAATAGGAACGTCTG TAATAGGAACGTCTG TAATAGGAACGTCT TAATAGGAACGTC TAATAG TAATAGGAACGTCTG TAATAGG (-6) TAATAGGAACG (-5) TAATAGGAACGTC TAATAGGAACGTCT TAATAGGAACGTCTG TAATAGGAACGTCT (-2) TAATAGGAACG TAATAGGAACG TAATAGGAACGTC TAATAGGAACG TAATAGGAACG TAATAGGAACG (-232) TAATAGGAACGTCTG TAATAGGAACGT TAATAGGAACGTC TAATAGGAACGTCTG TAATAGG TAATAGGAACGTC TAATAGGAACGTCTG TAATAGGAACGTCTG Metaphases with chromosome breaks 3 Metaphases with chromosome breaks Metaphases with chromosome breaks Metaphases with C C 205bp UR-GCACGGGGCC G GCACGGGGCCGTCGCC AAAC AACGTCTG T C G G T T T T G GG CAAA T T GACGTG G A GGGCGGACTGGGTGCT Untreated Metaphases with translocaTons Total aberrant metaphases 0 3 PD180970-treated and released CTACAATTCCG CAGCCTACAATTCCG G CCG ACAATTCCG CAGCCTACAATTCCG (-478) AGCCTACAATTCCG ATTCCG GCCTACAATTCCG (-232) (-2) CCG CAGCCTACAATTCCG GCCTACAATTCCG CAGCCTACAATTCCG CCG CAGCCTACAATTCCG CCTACAATTCCG CG ACAATTCCG TCCG TCCG ACAATTCCG CAGCCTACAATTCCG CAGCCTACAATTCCG (-303) CTACAATTCCG ATTCCG TCCG CAGCCTACAATTCCG CAGCCTACAATTCCG CAGCCTACAATTCCG AGCCTACAATTCCG CAGCCTACAATTCCG Total normal metaphases metaphases CTGGGGCCTCGGTGCA TCGGTGCA ACAACGTCTGTAGCGACC TAGCGACC CGCCCCCCCCCCT CCCCCCCCT AAGCCACGTGTA CACGTGTA ACAACCCCAGTGCCA Total analyzed % aberrant metaphases 202 205 Metaphases with translocaTons Total aberrant metaphases Total normal metaphases Total metaphases % aberrant translocaTons Total aberrant metaphases Total normal metaphases Total % aberrant metaphases metaphases analyzed metaphases analyzed 0 0 0 79 0.0 1.5 1 0 1 198 199 2 4 6 199 2.9 0 0 0 122 0.0 0.5 0 0 0 81 81 5 2 7 198 3.4 0 0 0 201 0.0 0 1 0 1 202 203 8 2 10 378 2.6 0 0 0 167 0.0 0.5 5 0 5 683 688 0.7 2 0 2 198 200 7 7 14 369 3.7 22 15 37 1144 3.1 1 0 1 116 0.9 0 0 0 310 0.0 1.0 0 0 0 79 79 3 4 7 203 3.3 1 0 1 593 0.2 0.0 5 0 5 384 389 4 9 13 187 6.5 0 0 0 108 0.0 1.3 5 0 5 95 100 5.0 12 0 12 756 768 12 7 19 364 5.0 8 6 14 86 14.0 1 0 1 304 0.3 1 0 1 134 0.7 1.6 2 4 6 214 220 27 26 53 840 5.9 2 0 2 546 0.4 2.7 2 0 2 164 166 7 8 15 185 7.5 0 0 0 141 0.0 1.2 4 4 8 378 386 4 2 6 186 1 0 1 99 1.0 3.1 2.1 2 0 2 208 210 11 10 21 371 0 1 1 219 0.5 5.4 1.0 0 0 0 200 200 0 5 5 405 0 0 0 300 0.0 1.2 0.0 ND ND ND ND ND ND 2 0 2 408 410 2 4 6 506 1.2 1 1 2 759 0.3 0 1 1 119 3 0 3 114 2.6 0.8 0.5 ND ND ND ND ND ND ND ND ND ND ND 2 10 12 1030 1.2 7 2 9 241 2 0 2 102 1.9 2 0 2 198 1.0 3.6 ND 4 4 8 122 130 6.2 4 4 8 122 130 8 8 16 235 6.4 7 0 7 414 1.7 7 13 20 224 3 0 3 143 2.1 8.2 6.2 ND ND ND ND ND ND 9 1 10 190 200 22 23 45 700 5 0 5 145 3.3 6.0 12 8 20 230 8 1 9 268 3.2 8.0 5 4 3 7 156 163 1 9 10 196 13 1 14 296 4.5 4.9 4.3 13 4 17 346 363 3 10 13 184 29 2 31 852 3.5 6.6 4.7 5 2 7 253 260 4 19 23 380 3 0 3 297 1.0 5.7 2.7 18 5 23 277 300 7.7 23 7 30 530 560 17 65 82 283 13 0 13 187 6.5 22.5 13 33 46 313 15 0 15 195 7.1 12.8 5.4 ND ND ND ND ND 30 98 128 596 7 0 7 193 3.5 17.7 ND 0 0 0 250 0.0 38 0 38 872 4.2
	35*				TAATAGGAACGTCTG	166bp UR-GGGCGGACTG	(-356)	CAGTGCCA
					TAATAGGAACG				AATTCCG
		ATTACCAACTTG ATTACC		(-18)	AA p value		Level	(-42)	ATAAGGCCGGTG GGCCGGTG
	Rag2 c/c XLF -/-p53 -/-	TAATAGGAACG vs XLF -/-p53 -/-	T 3.13e-9		***	TCCG
		ACCACCCCCACC vs Rag2 c/c p53 -/-	1.43e-4		***	GCTTCTTGAAGA
		ACCACCCC TACCAACTTGTC vs p53 -/-	(-168)	2.22e-12 p value	39bp inserTon *** Level	(-218)	CTTGAAGA CCGCCCCCCCCCCT
	Rag2 c/c XLF -/-Rag2 c/c XLF -/-p53 -/-	TACCAACT vs XLF -/-p53 -/-(-16) TAATAGGAACGTC vs XLF -/-3.92e-7 1.11e-5 TAATAGGAACGTC vs Rag2 c/c 6.71e-5 vs Rag2 c/c p53 -/-7.72e-9	G *** *** *** ***	(-1) CAGCCTACAATTCCG CAATTCCG	CCCCCCCCCT
		vs WT vs p53 -/-	TAATAGGAACGT TAATAGGAACG	C 8.55e-10 0.012	A * ***	AGCCTACAATTCCG AATTCCG
	XLF -/-XLF -/-p53 -/-	vs WT vs p53 -/-	TAATAGGAACG	0.16 0.021	T	*	ns	TCCG
	Rag2 c/c	GTCCGCCGACAC GTCCGCCG vs WT vs Rag2 c/c p53 -/-(-232)	0.29 0.539		ns ns	(-303)	AAGCCACGTGTA CACGTGTA
	Rag2 c/c p53 -/-	vs p53 -/-	TAATAGGAACG	0.077		ns	A	CCTACAATTCCG
					TAATAGGAACG				CAG	CAGCCTACAATTCCG
		GTCCGCCGACAC						AAGCCACGTGTA
		AGTCCTGC			(-179)				(-406)	CAAGGGGC
					TAATAGGAACG				A	CCTACAATTCCG
					TAATAGGAACG				CAGCCTACAATTCCG
					TAATAGGAACG		T		TCCG
					TAATAGGAACGTCTG			CAGCCTACAATTCCG
					TAATAGGAACG		T		TCCG
									CCTAACGTTACT
					TAATAGGAACGTCT			(-12)	ACGTTACT
					TAA		T		TCCG
					TAATAGGAACGTCTG			AGCCTACAATTCCG
		TGCTGACCACCCCCAC					GAACCCCCCACCTGGC
		TGCTGACC			(-168)		ACCCCC		(-264)	CACCTGGC
					TAATAGGAACGTCTG			AGCCTACAATTCCG
					TAATAGGAACG		T		TCCG
					TAATAGGAACGTCTG			AGCCTACAATTCCG
		GTCCGCCGACAC						AAGCCACGTGTA
		GTCCGCCG			(-232)				(-303)	CACGTGTA

v-abl pro-B cells. Number and percentage of aberrant metaphases harboring chromosomes breaks and/or translocaYons involving the Igk locus from untreated (a) and ABLki-treated and released (b) v-abl pro-B cell lines of the indicated genotype are indicated. DNA FISH experiments were performed using probes centromeric (Igk V) and telomeric (Igk C) to the Igk locus plus specific paint for chromosome 6. 
Rag2 c/c XLF -/-p53 -/-

  
	Expriment 1	17	9	26	218	10.7
	Expriment 2	23	7	30	259	10.4
	Expriment 3	15	2	17	193	8.1
	Expriment 4	19	5	24	176	12.0
	TOTAL	74	23	97	846	10.3

StaTsTcal analysis (Fisher exact test) Supplementary Table 4. Genomic instability at the Igh locus in primary pro-B cells.

  Number and percentage of aberrant metaphases harboring chromosomes breaks and/or translocaYons involving the Igh locus from cultured CD19 + IgM -pro-B cell of the indicated genotype are indicated. DNA FISH experiments were performed using probes centromeric (Igh V) and telomeric (Igh C) to the Igh locus plus specific paint for chromosome 12. 



In wild type lymphocytes (WT), the RAG proteins remain associated with coding ends in a post cleavage complex (RAG PCC), consYtuYng one Yer of the synapse (i.e. first safe-lock). The XLF-XRCC4 hetero-filaments appear to help tether broken coding ends and consYtute a second Yer of the synapse (i.e. second safe-lock). Core RAG2 does not exacerbate the phenotypes associated with ATM deficiency, suggesYng that the RAG complex and ATM act in the same pathway (i.e. the ATM-DDR/RAG PCC). When one safe-lock is missing (such as in Rag2 c/c , XLF -/-, Atm -/-, Rag2 c/c Atm -/-or Rag2 c/c cells treated with ATMki), overall DNA end repair is preserved but aberrant hybrid joint formaYon is observed, indicaYve of DNA end destabilizaYon. When both safe-locks are missing (such as in Rag2 c/c XLF -/-, XLF -/-ATM-DDR -/-, or XLF -/-cells treated with ATMki), collapse of the synapse leads to the release of DNA ends (which are potenYally subjected to ATM-dependent degradaYon), aborYon of V(D)J recombinaYon and genomic instability. We suggest that, in the context of normal physiological anYgen receptor assembly, the RAG1/2 endonuclease may have co-evolved with XLF to assist repair of generated DNA breaks through a very efficient double safe-lock mechanism, minimizing the inherent risks of genome instability and neoplasYc transformaYon.
Thymocytes number (x10 6