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. 2019 May 2;74(3):584-597.e9.
doi: 10.1016/j.molcel.2019.02.025. Epub 2019 Mar 21.

Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability

Christopher M Kirkham  1 , James N F Scott  1 , Xiaoling Wang  1 , Alastair L Smith  1 , Adam P Kupinski  1 , Anthony M Ford  2 , David R Westhead  1 , Peter G Stockley  1 , Roman Tuma  1 , Joan Boyes  3
Affiliations

Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability

Christopher M Kirkham et al. Mol Cell. .

Abstract

V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability.

Keywords: RAG proteins; V(D)J recombination; acute lymphoblastic leukemia; chromosome translocations; double strand breaks; genome instability.

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Figures

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Graphical abstract
Figure 1
Figure 1
SJ-RSS Cleavage Is Asymmetric (A) Cartoon of deletional V(D)J recombination and the generation of an ESC. (B) A SJ stimulates cleavage of 12- and 23-RSSs, but not vice versa. RAG cutting assays were performed using radiolabeled oligonucleotides, denoted by an asterisk above each set of lanes, carrying a 12-RSS, a 23-RSS, or a SJ sequence and separated on a native polyacrylamide gel. Unlabeled partner RSSs are present as indicated; S = SJ. Graphs represent mean of five experiments ± SD. (C) SJ-RSS cleavage is asymmetric at a range of core RAG concentrations. As for (B) except the amount of core RAG proteins was increased over an 8-fold range, indicated by the filled arrow; a 23-RSS partner was present in all reactions. (D) Cleavage reactions were performed as in (B) and uncut, nicked, and hairpinned (HP) DNA was separated on a denaturing gel. nt, nucleotides. Graphs represent mean of three experiments ± SD. See also Figures S1 and S2 and Table S1.
Figure 2
Figure 2
RAGs Bind to Both RSSs in the SJ (A) A slower migrating complex is formed with the SJ. RAG complexes were formed with labeled oligonucleotides carrying a 12-RSS (lanes 3–8), 23-RSS (lanes 10–14), or SJ (lanes 16–20) and run on a 4% polyacrylamide gel. Increasing amounts of unlabeled partner RSSs are present as indicated above the gel. When the SJ is labeled, a third complex of higher molecular weight is visible (indicated by “C”). This is not visible in lanes containing unlabeled SJ, most likely because the paired complex has an average lifetime of ∼400 s (Lovely et al., 2015) and would dissociate before loading on the gel. SC1/2, single complex 1/2; HSC1/2, HMGB1 single complex 1/2; PC, paired complex. (B) DNaseI footprint shows both RSSs are occupied. The wild-type (WT) SJ is on the left and the position of the RSSs, as determined from the G+A marker ladder is shown to the right of this gel. Triangles indicate nonamer sequences and pentamers represent hexamers. Bars indicate protected regions; stars depict enhanced cutting. Footprints using SJ probes where the 12-RSS, 23-RSS, or both RSSs are mutated in heptamer and nonamer sequences are shown to the right. Protection of mutated heptamers adjacent to the WT RSS with SJd12 and SJd23 likely results from RAG binding to flanking DNA (Nagawa et al., 2002, Nagawa et al., 2004).
Figure 3
Figure 3
Mutating One RSS within the SJ Restores Symmetric Cleavage (A) The 23-RSS nonamer within the SJ was deleted and the oligonucleotide used in cutting reactions either as a partner for labeled 12- or 23-RSSs or as the substrate. Symmetric cutting was almost restored when SJ23d9 was paired with a 23-RSS. (B) Deletion of the nonamer within the 12-RSS of the SJ results in more symmetric cutting with a 12-RSS. As for (A) except the oligonucleotide SJ12d9 was used. (C and D) RAG binding to mutant SJs. RAG complexes were formed with the 12-RSS, wild-type SJ, or mutant SJs and resolved on a native gel. Complex “C” is formed with the wild-type SJ but is reduced with SJ23d9 and SJ12d9. This is most pronounced in the presence of a partner RSS that likely outcompetes weaker RAG binding to the mutant SJ. See also Figure S3.
Figure 4
Figure 4
The ESC Poses a Risk to Genome Stability (A) The SJ triggers cutting at cRSSs. RAG cutting assays were performed using radiolabeled oligonucleotides, denoted by an asterisk, carrying a 12-RSS or the LMO2 cRSS. Unlabeled 23-RSS or SJ partners are present as indicated. (B) Signal ends are released faster following a SJ-RSS cutting reaction than a 12/23-RSS reaction. Cutting reactions, using oligonucleotides labeled on the lower strand to detect signal ends, were performed at 37°C, followed by incubation at the temperatures indicated. The reaction was divided into two and resolved on a native gel (top) and a cutting gel (bottom). Release of the signal ends (indicated by the arrow, top gel) was calculated relative to the level of cutting (bottom gel). Data obtained with core RAG1 and full-length RAG2 are shown; equivalent data were obtained with core RAG2. nR = no RAGs. Data are represented as mean of three experiments ± SD. Increased SE release in the presence of the SJ is statistically significant (Student’s t test; ∗p = 0.01 [37°C] and ∗∗∗p = 0.0001 [42°C]). See also Figure S4.
Figure 5
Figure 5
Cleavage of SJ-RSS Substrates Is Asymmetric In Vivo (A) REH cells were transduced with integrase-deficient lentiviruses carrying a 12-RSS, a 23-RSS, or a wild-type or mutant SJ sequence, as indicated. The amount of intact substrate after 48 h was measured by qPCR. Data were normalized to unique regions within each provirus, and the values given are relative to controls where lentiviruses carrying a partner sequence were not transduced. Data are represented as mean of three separate transductions ± SD. See also Figure S5. (B) LM-PCR was performed using the samples from (A), and the products are shown under the respective graphs. The template amplified by LM-PCR is indicated above the lanes with an asterisk. Ligation of the linker primer to a RAG cleaved end recreates an ApaLI site and digestion was used to verify RAG-mediated cutting. For the SJ mutations (and SJ), the RSS that is expected to be blunt following cleavage was amplified by LM-PCR. Amplification of each of the SJ ends is in the quantitative range and since the same primers were used to amplify the SJ-12-RSS end and SJ23d7, the increased signal with SJ23d7 indicates increased cutting, likewise for the SJ-23-RSS end and SJ12d9. SJ12d7 and SJ23d9 were cloned into the vector in the reverse orientation and gave different sized products with some primers. This also resulted in cross-reactivity of SJ23d9 with 23-RSS primers, giving an additional band (right gel, lane 13). Samples were normalized using qPCR; the bottom (PCR) shows PCRs with the normalization primers to verify the respective vectors were present. (C) Unligated signal ends are present in vivo. Top: cartoon of the Igλ locus. Bottom: DNA from pro-B cells of PIP3 transgenic mice, where the Igλ locus undergoes premature recombination in pro-B cells, was used in an LM-PCR reaction to amplify Vλ1 and Jλ1 signal ends. ApaLI was used to cleave the signal ends prior to LM-PCR to give a positive control for unligated signal ends. No ligase controls are shown; C indicates the no template control. (D) The majority of signal ends are ligated. The signal joints and coding joints of three distinct ESCs or recombination products were quantified against a standard curve of known numbers of copies and the relative amounts calculated. Data are represented as mean of three experiments ± SD.
Figure 6
Figure 6
The SJ Triggers DSB In Vivo (A) Left: γH2AX foci in Cos7 cells transfected with RAG expression vectors and plasmids carrying the 12- or 23-RSS or SJ12d9 or SJ. One representative example of a vector alone transfection is shown. Middle, top graph: quantification of γH2AX foci from 450 Cos7 cells per transfection condition and three independent experiments. (B) REH cells were transduced with EGFP-expressing integrase-deficient lentiviruses, that carry no additional sequence (control virus), the 23-RSS, SJ12d9, or the SJ sequence. γH2AX foci were detected using an AlexaFluor568-labeled (red) secondary antibody. Middle, bottom graph: foci in 450 REH cells per condition from three independent experiments. Fewer foci are observed than in (A) because only 3–6 RSS or SJ vectors are present per cell. Error bars show SD.
Figure 7
Figure 7
The SJ Causes Chromosome Breaks (A) Apoptosis in transduced REH cells as measured by annexin V and propidium iodide (PI) staining for early and late stage apoptosis, respectively. Data are represented as mean of three independent experiments ± SD; increased early stage apoptosis in the presence of the SJ compared to the other vectors is statistically significant (Student’s t test; ∗∗∗p = 0.0002 [control vector], ∗∗p = 0.0064 [23-RSS], ∗p = 0.011 [SJ12d9]). (B) Example of two SJ-mediated breaks that co-localize with breaks found in ALL patients. Chromosome breaks mapping close to the cRSS at 194697727 on chromosome 1 were observed in six ALL patients and in two independent LAM-HTGTS experiments. Similar co-localization was observed for 22 more patients (see also Figures S6, S7, and Table S2). (C–E) Graphs showing the frequency of breaks in the 13 genes that most frequently acquire somatic mutations in ETV6/RUNX1-positive ALL (Papaemmanuil et al., 2014) in the presence of the SJ compared to the 12-RSS (C), 23-RSS (D), and SJ12d9 (E), respectively. (F) As for (E) but comparing the frequency of breaks in genes that are commonly mutated in all types of B-ALL. See also Table S3.
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