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V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells
Introduction
Materials And Methods
Cell culture
Transfections
Assays for signal joints and signal ends
Assay for coding ends and coding joints
Assays for hybrid joints and inversional signal joints
Results
Experimental design
Abundant, full-length signal ends in XRCC4-deficient cells
Abundant coding ends in XRCC4-deficient cells
Efficient formation of hybrid joints in XRCC4-deficient cells
Nucleotide sequence analysis of hybrid joints
Discussion
References
V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells
ABSTRACT
INTRODUCTION
In order to generate diversity of immunoglobulin (Ig) and T cell receptor (TCR) molecules, the vertebrate immune system utilizes a site-specific DNA rearrangement process termed V(D)J recombination which assembles the variable regions of Ig and TCR genes from multiple germline V, (D) and J gene segments (1,2). The V(D)J recombination machinery recognizes short DNA sequence elements, termed recombination signal sequences (RSSs), that are found adjacent to germline V, (D) and J coding segments. An RSS consists of conserved heptamer and nonamer elements separated by non-conserved spacer sequences of 12 or 23 nucleotides. Recombination is initiated by introduction of double strand breaks (DSBs) precisely at the border between the heptamer and the first nucleotide of the coding segment, generating two types of DNA termini: hairpin coding ends and blunt signal ends (Fig.
Figure 1. Overview of V(D)J recombination and analysis of signal joints. (A) Overview of the V(D)J recombination reaction. The plasmid recombination substrate pJH290 is illustrated. Cleavage at the RSS (open triangle, 12-signal; filled triangle, 23-signal) produces an excised fragment terminating in signal ends and a pair of hairpin coding ends on the plasmid backbone. Joining of these ends produces an excised circular molecule containing a signal joint and a coding joint on the plasmid. (B) Analysis of signal joints. Signal joints were amplified by PCR (24 cycles) using DR55 and ML68 primers. DNA amounts added to PCR reactions are indicated above each lane. `1X' indicates 2% of the DNA recovered from the each transfection; `1:10', `1:100', etc. indicate the dilution used for PCR amplification. `+R' and `-R' denote the presence or absence of the RAG-1/RAG-2 expression vectors in the transfection. Marker lane (M) contains radiolabeled 1 kb ladder (Gibco-BRL). Numbers to the left of the gel are marker (M) sizes in bp. The blot was hybridized with an end-labeled oligonucleotide probe (DR55). The recombination signals are symbolized as open (12-spacer signal) or filled (23-spacer signal) triangles. The vertical arrows represent the sites of cleavage. Curved arrows indicate PCR primers. Cleavage at the RSS is performed by RAG-1 and RAG-2 proteins (8) that are expressed specifically in developing lymphocytes. In contrast, the joining of the broken DNA ends is carried out by a number of proteins that are expressed in many cell types and are also essential for repair of DSBs. So far, four genes are known to encode proteins involved in both DSB repair and V(D)J recombination (9-12). Three of these genes (XRCC5, XRCC6 and XRCC7) encode components of the DNA-dependent protein kinase (DNA-PK) (13,14). DNA-PK is a nuclear serine-threonine protein kinase and consists of a catalytic subunit, DNA-PKcs, and a heterodimeric DNA end binding component, Ku, which is made up of 70 and 86 kDa subunits. The kinase activity of DNA-PK is stimulated by binding to DNA ends, and it has been suggested that DNA-PK may serve to signal the presence of DNA damage through phosphorylation of target proteins (14,15). DNA-PK may also play a more direct role in repair of damage, for example by serving as a scaffold for assembly of a repair complex (14,15). The role of XRCC4, the fourth factor identified by analysis of DSB repair mutants, remains enigmatic. The XRCC4-deficient Chinese hamster ovary (CHO) cell line, XR-1, is hypersensitive to ionizing radiation (16), and is severely defective for V(D)J recombination (11,17). Although XRCC4 has been cloned, the predicted amino acid sequence of the protein provides no information about its function (17). Several lines of evidence indicate that the XRCC4 protein is not an essential component of DNA-PK (14,17-19). XRCC4 is a substrate for DNA-PK in vitro (20,21); however, the physiological relevance of this observation is unclear because phosphorylation is not required for XRCC4 function (21). Recent work has shown that XRCC4 binds to DNA ligase IV and stimulates its activity in vitro (20,22), indicating that XRCC4 may function to stimulate ligation of broken DNA molecules. However, XRCC4 may have other functions, and the precise roles of XRCC4 in V(D)J recombination and DSB repair remain unclear. Analysis of the rare coding (11) and signal joints (11,17) isolated from XRCC4-deficient cells revealed excessive loss of nucleotides, providing one clue to the function of XRCC4 in V(D)J recombination. Based on these data it was proposed that in the absence of XRCC4, V(D)J recombination intermediates are hypersensitive to degradation, so that ends rarely survive long enough to undergo joining; hence, those joints that are formed often contain large deletions (11,17). This `end protection' model predicts that broken DNA recombination intermediates would undergo excessive degradation in XRCC4-deficient cells. Our laboratory has developed methods that allow direct examination of broken ends derived from cleavage of extra-chromosomal V(D)J recombination substrates transiently transfected into fibroblasts along with expression vectors encoding RAG-1 and RAG-2 (23,24). Here we have used this system to examine signal and coding ends created during V(D)J recombination of plasmid substrates in XRCC4-deficient cells. Our results show that these cells contain abundant full-length signal ends and abundant hairpin coding ends, providing the first direct evidence that recombination intermediates are not subjected to excessive degradation in the absence of XRCC4. Furthermore, we find that non-standard V(D)J recombination products, termed hybrid joints, which involve joining of signal ends to coding ends, form efficiently in XRCC4-deficient cells. In addition, most hybrid joints formed in the absence of XRCC4 do not show excessive deletion. Together these data indicate that impaired formation of standard V(D)J recombination products in XRCC4-deficient cells does not result from excessive degradation of DNA ends.
MATERIALS AND METHODS
Cell culture
The cell lines used in this study (CHOK1 4364, RMP41, XR-1 and XR-1 TR) have been described previously (16,25,26, unpublished observations). Cells were maintained in Dulbecco's modified Eagle's medium enriched with 10% fetal bovine serum and 0.1 mM MEM non-essential amino acids as described previously (24).
Transfections
Transfections were done using a lipofection reagent (Gibco-BRL) according to the manufacturer's protocol. Two micrograms each of the recombination substrate and truncated (core) RAG-1 (pMS127b)/RAG-2 (pMS216) expression vectors (27,28) were co-transfected into cells. Although the results shown here were obtained with truncated RAG proteins, similar data were generated using expression vectors encoding the full-length RAG proteins. DNA was recovered after 48 h using the method of Hirt as described previously (23). Transfection efficiencies were determined using an assay which identifies cells that have been transfected with plasmids encoding [beta]-galactosidase (29).
Assays for signal joints and signal ends
Signal joints were detected by circular PCR (24 cycles) using DR55 and ML68 primers as described previously (24). Signal ends were detected by ligation-mediated PCR (LMPCR) using the DR55 primer for 12-signal end and the ML68 primer for 23-signal end, respectively, as described previously (5,23). ApaL1 digestion and T4 DNA polymerase treatments were performed as described (5,30). PCR products were separated on 6% polyacrylamide gels, and probed with end-labeled oligonucleotide probes (DR73, DR69, ML68 and DR55), or to an internally labeled PvuII fragment of pJH290 as described (24,30). Excised linear recombination intermediates were assayed by Southern blotting as described (24).
Assay for coding ends and coding joints
Excised linear recombination intermediates containing coding end(s) were assayed by Southern blotting as described (24). Hairpin coding ends were detected by LMPCR using ML68 and DR20 primers (30). Mung bean nuclease pretreatment was performed as described (7). Coding joints were detected by PCR using DR55 and ML68 primers (24 cycles of amplification).
Assays for hybrid joints and inversional signal joints
To detect hybrid joints, DNA samples were amplified by PCR using the DR55 and ML68 primers described above. Amplified hybrid joints were detected by hybridization with hybrid junction-specific (DR98) or non-junction-specific (DR55) oligonucleotide probes as described (30). Inversional signal joints were detected by PCR with DR55 and DR100 primers (30).
RESULTS
Experimental design
Substrates were introduced into each of four different CHO fibroblast cell lines: XR-1 (XRCC4-deficient), XR-1 TR (XR-1 complemented with a wild-type XRCC4 cDNA), the parental wild-type cell line (CHOK1 4364; hereafter designated CHOK1), or a related wild-type cell line, RMP41. In addition to the recombination substrates, expression vectors encoding RAG-1 and RAG-2 were transfected, as described previously (23,27). DNA was recovered after 48 h and signal ends and V(D)J recombination products were detected using semiquantitative PCR amplification or Southern blotting, as described previously (23,24,30). Amplified products were analyzed by polyacrylamide gel electrophoresis followed by hybridization to a radiolabeled probe. All transfections were repeated at least three times with similar results.
Abundant, full-length signal ends in XRCC4-deficient cells
A sensitive PCR assay was used to detect signal joints. As expected, signal joints were readily detected in wild-type cells, even using a 1:100 dilution of DNA recovered from the transfection (Fig.
Two observations have led to the suggestion that XRCC4 might be required for protection of the broken end intermediates: the severely decreased abundance of signal joints in XRCC4-deficient cells and the presence of aberrant deletions at those signal joints which are recovered (11,17). Therefore, we examined the signal ends using an LMPCR assay we have employed previously for this purpose (23,30). Analysis of signal ends derived from cleavage at the RSS containing a 23 nt spacer (23-signal) is shown in Figure
Figure 2. Analysis of signal ends by LMPCR. Blunt signal ends were detected by LMPCR. Products derived from pJH290 are of the expected sizes (arrows to the left of the gel). The amounts of DNA added to each ligation are indicated. Five percent of the ligated DNA was used for PCR reactions. Blots were probed with the DR69 oligonucleotide as described (23). Arrows represent PCR primers; a pair of heavy lines denotes ligation primers. (A) Analysis of 23-spacer signal ends derived from pJH290. (B) Analysis of 12-spacer signal ends derived from pJH290. To determine whether the signal ends were subject to nucleotide loss, additional studies were performed. Since only a full-length, blunt signal end generates an ApaL1 restriction site upon ligation with the primer (Fig. Figure 3. Analysis of the structure of signal ends. (A) ApaL1 digestion assay. A full-length, blunt signal end generates an ApaL1 restriction site (5[prime]-GTGCAC) upon ligation with a double-stranded oligonucleotide DR20, as described previously (5). (B) Analysis of 23-signal ends. LMPCR products derived from the 23-signal end were subjected to digestion with ApaL1. Undigested (`-Apa') and digested (`+Apa') samples were loaded in adjacent lanes. Blots were probed with a PvuII fragment from pJH290. Similar results were obtained for 12-signal ends (data not shown). (C) Pretreatment with T4 DNA polymerase. To detect non-blunt 23-signal ends (left panel) or 12-signal ends (middle panel), DNA samples were treated with T4 DNA polymerase (Gibco-BRL) prior to ligation as described (7). +T4 and -T4 indicate the presence and absence of T4 DNA polymerase in the reactions. Blots were probed with a 32P-labeled PvuII fragment from pJH290. As a control for the activity of T4 DNA polymerase (right panel), BALB/c testis DNA was digested with PstI to produce ends with 3[prime] extensions, which were then subjected to LMPCR with and without T4 DNA polymerase pretreament as described previously; the blot was probed with a TCR[delta]-specific probe (7). To search for signal ends that might terminate in short single-stranded extensions resulting from loss of nucleotides from either 5[prime] or 3[prime] termini, DNA samples were treated with T4 DNA polymerase prior to LMPCR to convert potential non-blunt ends to blunt form (7). As shown in Figure One potential explanation for the presence of abundant signal ends in the absence of detectable signal joints in XRCC4-deficient cells is that cleavage does not occur at both signal sequences. To address this possibility, Southern blotting was performed to measure dual RSS cleavage (Fig. Figure 4. Southern blotting analysis for excised linear (double cleaved) intermediates. The indicated cell lines were transfected with pJH290 in the presence (`+R') or in the absence (`-R') of RAG-1 and RAG-2. Twenty percent of the DNA recovered from each transfection was analyzed by Southern blotting. Blots were probed with the ML68 oligonucleotide. We next examined coding ends generated in XRCC4-deficient cells. As shown in Figure Figure 5. Analysis of coding ends and coding joints. (A and B) Southern blotting analysis for coding ends. Twenty percent of the DNA recovered from each transfection with pJH289 (A) and pJH299 (B) in the presence (`+R') or in the absence (`-R') of the RAG-1/RAG-2 expression vectors was analyzed by Southern blotting. Blots were probed with the ML68 oligonucleotide. (C) Analysis of the structure of coding ends. To detect hairpin coding ends, DNA samples (1 µl of undiluted DNA) were pre-treated with mung bean nuclease (Gibco-BRL) prior to ligation (7). +M and -M indicate the presence and absence of mung bean nuclease in the reactions. Blots were probed with the oligonucleotide, DR82 (5[prime]-ATGAGAGGATCCCACGAATTCCCG-3[prime]). Similar results were obtained using a PvuII fragment from pJH290 as probe (data not shown). (D) Analysis of coding joints. Coding joints were detected by PCR (24 cycles) using DR55 and ML68 primers. The amounts of DNA added to each PCR are indicated above each lane. `+R' and `-R' indicate the presence or absence of the RAG-1/RAG-2 expression vectors in the transfection. The blot was hybridized with an end-labeled DR55 oligonucleotide. Similar results were obtained with probing with a PvuII fragment from pJH290 (data not shown). To analyze the structure of the coding ends, an LMPCR-based assay was performed. DNA samples were pretreated with mung bean nuclease prior to LMPCR to convert hairpin ends to blunt ends which can then be ligated to blunt ligation primers (7). As shown in Figure The detection of hairpin coding ends in wild-type cells was surprising because hairpin coding ends were not detected in our previous analysis of wild-type mice (7). We have considered two potential explanations for this result. First, the overexpression of RAG-1 and RAG-2 proteins in our transient transfection system may stabilize the hairpin coding ends. This is supported by the recent observation that all four DNA ends (the coding and signal ends) form a stable complex with Rag proteins after the cleavage reaction (31). Second, the extremely high levels of cleavage observed in our transient transfection system may outstrip the capacity of the cells to process the broken ends, leading to the accumulation of a population of hairpin coding ends. Because we observed that the abundance of hairpin coding ends is not affected by the absence of XRCC4, we wanted to confirm whether joining of these ends is indeed impaired in XRCC4-deficient cells. PCR analysis failed to reveal coding joints in XR-1 cells even when undiluted (1X) DNA samples were used (Fig. The experiments shown above demonstrated that intact signal ends and coding ends are present in XRCC4-deficient cells. We then asked whether these ends might be available for some other joining reactions, such as formation of non-standard products, termed hybrid joints (32,33). Hybrid joints result from joining a coding end to a signal end (diagrammed in Fig. Figure 6. Efficient formation of hybrid joints in XR-1 cells. (A) Standard and non-standard junctions derived from an inversion substrate. The recombination substrate, pJH299, is diagrammed. The standard products, coding and signal joints, are formed by inversion, as shown on the right. Alternatively, recombination can produce non-standard junctions (hybrid joints) by deletion, as shown on the left. Arrows indicate PCR primers used to detect recombinant junctions. (B) No inversions are detected in XR-1 cells. Inversional signal joints generated from pJH299 were detected by PCR using DR100 and DR55 as PCR primers. Blots were probed with the DR55 oligonucleotide. The signal ends on the plasmid were detected by LMPCR using DR100 and DR20 PCR primers (30). PCR products were detected by hybridization with the DR69 oligonucleotide. (C) Hybrid joints are formed efficiently in XR-1 cells. Cleavage of pJH299 produces both signal and coding ends on the excised products. The joining of the ends generates hybrid joints on the excised product. Hybrid joints were amplified by PCR using DR55 and ML68 primers and probed with the hybrid junction-specific oligonucleotide probe (DR98) (30). DR55 and DR20 were used as LMPCR primers for 12-spacer signal ends on the excised product. The blot was probed with DR69. To examine the structure of hybrid joints in detail, PCR products were cloned and sequenced. The results (Fig. Figure 7. Nucleotide sequence analysis of hybrid joints. PCR products containing hybrid joints were cloned and sequenced. The solid triangle indicates the position of the junction. * indicates that the end from which the deletion occurred cannot be assigned due to a 1 nt homology (deletions were arbitrarily assigned to the coding end). Letters at the junctions indicate extra nucleotides. Circled letters indicate presumptive P nucleotides. `n' indicates the number of junctions with the indicated sequence. Some junction sequences from CHOK1 cells were reported previously (30). The number of nucleotides deleted from each end is indicated. Although the majority of junctions isolated from XR-1 cells retain all nucleotides of both ends, some deletions from coding and signal ends were observed. Most of these deletions (four out of five junctions) fell within the range of deletion lengths (up to 16 nt from the coding end and up to 31 nt from the signal end) observed in hybrid joints from the control cell lines. A single junction from XR-1 cells showed large deletions from both coding (-24) and signal ends (-57). This junction, as well as two other junctions with deletions, contained a short sequence homology. However, short sequence homologies were not universal features of junctions formed in XR-1 cells, as hybrid joints both with and without deletions were recovered that lack junctional homology. Together, these data indicate that there is no general tendency toward excessive deletions at hybrid joints formed in XR-1 cells. Four junctions from XR-1 cells contained single extra G residues that can be explained as P nucleotides derived from hairpin opening (30,34,35). Such insertions were also seen in hybrid joints formed in the XR-1 TR and CHOK1 cell lines (Fig. Our analysis of V(D)J recombination intermediates in XRCC4-deficient cells revealed abundant, full-length signal ends and hairpin coding ends. We also found that hybrid joints are formed efficiently in these cells, without excessive deletion. These results indicate that XRCC4 is not required to protect the broken DNA intermediates produced during V(D)J recombination from excessive degradation. Instead, we suggest that the ends are protected by persistence of a stable post-cleavage complex containing the Rag proteins which does not require the presence of XRCC4. This view is supported by the observation that purified RAG-1 and RAG-2 form a complex with recombination signal sequences (36); furthermore, stable complexes containing signal ends and Rag proteins have been detected in vitro (31,37). Although others have found that hybrid joints can be formed from artificial substrates stably integrated into chromosomal DNA of XRCC4-deficient cells (17), the relative abundance of these products was not measured and nucleotide sequences of the junctions were not reported. Here we show that the absence of XRCC4 does not affect the abundance of hybrid joints (measured relative to levels of signal ends in the same DNA samples). Thus, while joining of a signal end to a signal end, or a coding end to a coding end, is decreased by at least 50-100-fold (13,17, this work), joining of a coding end to a signal end proceeds with normal efficiency in the absence of XRCC4. What are the mechanisms responsible for efficient formation of hybrid joints in XRCC4-deficient cells? We recently found that hybrid joints form efficiently and without excessive nucleotide loss in Ku86-deficient mice (38) and cell lines (30). To explain these results, we hypothesized that the Rag proteins might be capable of joining a signal end to a coding end, a reaction we have termed Rag-mediated joining. This could occur by reversing the second step of the cleavage reaction, using the 3[prime] OH of the signal end to attack the hairpin coding end, forming a hybrid joint which retains all nucleotides of both the coding and signal ends (30,38). This proposal is supported by recent experiments showing that purified Rag proteins are capable of forming hybrid joints from artificial substrates (39). These joints can be perfect, with no additional nucleotides; however, opening of the hairpin away from the axis of symmetry (the `tip' of the hairpin) would allow the incorporation of P nucleotide insertions (30,38,39). Thus, Rag-mediated joining could account for most hybrid joints formed in XRCC4-deficient cells. Our data suggest that, rather than playing an indirect role in joining (such as protecting DNA ends), XRCC4 instead may participate directly in end joining. This hypothesis is consistent with recent work showing that XRCC4 interacts with and stimulates the activity of DNA ligase IV in vitro (22). The proposal that XRCC4 functions to stimulate ligase activity could explain the invariable presence of short sequence homologies (microhomologies) at signal joints isolated from XRCC4-deficient cells (17). This feature, which is not observed at signal joints from wild-type cells, suggests that in the absence of XRCC4, formation of these junctions is dependent upon base pairing interactions provided by microhomologies. Recent work has shown that Ku stimulates the activity of all three mammalian DNA ligases in vitro, and reduces the requirement for complementary overhangs (40). XRCC4 may be stimulating ligation in an analogous fashion, allowing signal joints to form by blunt-end ligation, without a requirement for base pairing. However, the rather modest degree of stimulation of DNA ligase IV activity seen in vitro upon addition of XRCC4 (<10-fold) may not be sufficient to explain the much more severe defects (100-fold) in coding and signal joint formation observed in XRCC4-deficient cells. Furthermore, we have recently shown that joining of DNA ends created by restriction enzyme cleavage is not severely impaired in these cells, indicating that the basic enzymatic machinery required for end joining is intact (unpublished observations). These observations suggest that XRCC4 may play critical roles in V(D)J recombination in addition to interacting with ligase IV. This hypothesis is supported by recent observations indicating that XRCC4 facilitates binding of Ku to DNA and complexes with DNA-bound DNA-PK (21). Potential functions of XRCC4 include direct roles in end joining, such as alignment of DNA termini or recruitment of additional joining or processing activities. Additionally or alternatively, XRCC4 could have indirect roles such as disassembly of post-cleavage RAG-DNA complexes to make the ends available for joining, as we have suggested previously for components of DNA-PK (41). According to this view, the severe defects in formation of standard V(D)J recombination products in XRCC4-deficient cells are caused by a failure in the joining reaction (which can be overcome in the case of hybrid joints, if RAG proteins perform joining), rather than by a failure of end protection. How does this model explain the excessive deletions observed at coding and signal joints from XRCC4-deficient cells? The rare joints formed in the absence of XRCC4 may arise by alternative pathways that promote loss of nucleotides. For example, defective release of ends from post-cleavage complexes could be bypassed by rare endonucleolytic cleavage events which generate free ends, promoting joining accompanied by deletion. Similar mechanisms have been suggested to explain deletions at junctions formed in Ku-deficient cells (30,41).
Abundant coding ends in XRCC4-deficient cells
Efficient formation of hybrid joints in XRCC4-deficient cells
Nucleotide sequence analysis of hybrid joints
DISCUSSION
REFERENCES
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