Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase

V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.     

Modulation of terminal deoxynucleotidyltransferase activity by the DNA-dependent protein kinase.

Rare Ig and TCR coding joints can be isolated from mice that have a targeted deletion in the gene encoding the 86-kDa subunit of the Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). However in the coding joints isolated from Ku86-/- animals, there is an extreme paucity of N regions (the random nucleotides added during V(D)J recombination by the enzyme TdT). This finding is consistent with a decreased frequency of coding joints containing N regions isolated from C.B-17 SCID mice that express a truncated form of the catalytic subunit of the DNA-PK (DNA-PKCS). This finding suggests an unexpected role for DNA-PK in addition of N nucleotides to coding ends during V(D)J recombination. In this report, we establish that TdT forms a stable complex with DNA-PK. Furthermore, we show that DNA-PK modulates TdT activity in vitro by limiting both the length and composition of nucleotide additions.     

Terminal deoxynucleotidyl transferase requires KU80 and XRCC4 to promote N-addition at non-V(D)J chromosomal breaks in non-lymphoid cells

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that increases the repertoire of antigen receptors by adding non-templated nucleotides (N-addition) to V(D)J recombination junctions. Despite extensive in vitro studies on TdT catalytic activity, the partners of TdT that enable N-addition remain to be defined. Using an intrachromosomal substrate, we show here that, in Chinese hamter ovary (CHO) cells, ectopic expression of TdT efficiently promotes N-additions at the junction of chromosomal double-strand breaks (DSBs) generated by the meganuclease I-SceI and that the size of the N-additions is comparable with that at V(D)J junctions. Importantly, no N-addition was observed in KU80- or XRCC4-deficient cells. These data show that, in a chromosomal context of non-lymphoid cells, TdT is actually able to promote N-addition at non-V(D)J DSBs, through a process that strictly requires the components of the canonical non-homologous end-joining pathway, KU80 and XRCC4.     

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining phase of the V(D)J recombination reaction.     

Evidence that the long murine terminal deoxynucleotidyltransferase isoform plays no role in the control of V(D)J junctional diversity

Two TdT isoforms have been found in the mouse. The short isoform is known to add N regions to gene segment junctions during V(D)J recombination, but the role of the long (TdTL) isoform is controversial. We have shown that TdTL, although endowed with terminal transferase activity, is thermally unstable and unable to add N regions in vivo. In this study, we demonstrate that TdTL is devoid of 3'-5' exonuclease activity, and provide an analysis of nucleotide deletion and addition patterns in large series of V(D)J coding joins, arguing against a role of TdTL in the control of junctional diversity in Igs and TCRs.     

V(D)J recombination: site-specific cleavage and repair

V(D)J recombination is a site-specific gene rearrangement process that contributes to the diversity of antigen receptor repertoires. Two lymphoid-specific proteins, RAG1 and RAG2, initiate this process at two recombination signal sequences. Due to the recent development of an in vitro assay for V(D)J cleavage, the mechanism of cleavage has been elucidated clearly. The RAG complex recognizes a recombination signal sequence, makes a nick at the border between signal and coding sequence, and carries out a transesterification reaction, resulting in the production of a hairpin structure at the coding sequence and DNA double-strand breaks at the signal ends. RAG1 possesses the active site of the V(D)J recombinase although RAG2 is essential for signal binding and cleavage. After DNA cleavage by the RAG complex, the broken DNA ends are rejoined by the coordinated action of DNA double-strand break repair proteins as well as the RAG complex. The junctional variability resulting from imprecise joining of the coding sequences contributes additional diversity to the antigen receptors.     

Non-consensus heptamer sequences destabilize the RAG post-cleavage complex,making ends available to alternative DNA repair pathways

V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC’s ability to restrict repair pathway choice.     

Mechanistic constraints on diversity in human V(D)J recombination.

We have analyzed a large collection of coding junctions generated in human cells. From this analysis, we infer the following about nucleotide processing at coding joints in human cells. First, the pattern of nucleotide loss from coding ends is influenced by the base composition of the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences. Second, inverted repeats can occur at ends that have undergone nucleolytic processing. Previously, inverted repeats (P nucleotides) have been noted only at coding ends that have not undergone nucleolytic processing, this observation being the basis for a model in which a hairpin intermediate is formed at the coding ends early in the reaction. Here, inverted repeats at processed coding ends were present at approximately twice the number of junctions as P nucleotide additions. Terminal deoxynucleotidyl transferase (TdT) is required for the appearance of the inverted repeats at processed ends (but not full-length coding ends), yet statistical analysis shows that it is virtually impossible for the inverted repeats to be polymerized by TdT. Third, TdT additions are not random. It has long been noted that TdT has a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highly significant frequency. This tendency suggests that nucleotide-stacking interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D)J recombination.     

In vivo transposition mediated by V(D)J recombinase in human T lymphocytes

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.     

A biochemically defined system for coding joint formation in V(D)J recombination

V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.     

Hybrid joint formation in human V(D)J recombination requires nonhomologous DNA end joining

In V(D)J recombination, the RAG proteins bind at a pair of signal sequences adjacent to the V, D, or J coding regions and cleave the DNA, resulting in two signal ends and two hairpinned coding ends. The two coding ends are joined to form a coding joint, and the two signal ends are joined to form a signal joint; this joining is done by the nonhomologous DNA end joining (NHEJ) pathway. A recombinational alternative in which a signal end is recombined with a coding end can also occur in a small percentage of the V(D)J recombination events in murine and human cells, and these are called hybrids (or hybrid joints). Two mechanisms have been proposed for the formation of these hybrids. One mechanism is via NHEJ, after initial cutting by RAGs. The second mechanism does not rely on NHEJ, but rather invokes that the RAGs can catalyze joining of the signal to the hairpinned coding end, by using the 3'OH of the signal end as a nucleophile to attack the phosphodiester bonds of the hairpinned coding end. In the present study, we addressed the question of which type of hybrid joining occurs in a physiological environment, where standard V(D)J recombination presumably occurs and normal RAG proteins are endogenously expressed. We find that all hybrids in vivo require DNA ligase IV in human cells, which is the final component of the NHEJ pathway. Hence, hybrid joints rely on NHEJ rather than on the RAG complex for joining.     

DNA Hairpin Opening Mediated by the RAG1 and RAG2 Proteins

The lymphoid cell-specific proteins RAG1 and RAG2 initiate V(D)J recombination by cleaving DNA adjacent to recombination signals, generating blunt signal ends and covalently sealed, hairpin coding ends. A critical next step in the reaction is opening of the hairpins, but the factor(s) responsible has not been identified and had been thought to be a ubiquitous component(s) of the DNA repair machinery. Here we demonstrate that RAG1 and RAG2 possess an intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip. In Mn2+, a synthetic hairpin is nicked 5 nucleotides (nt) 5' of the hairpin tip, with more distant sites of nicking suppressed by HMG2. In Mg2+, hairpins generated by V(D)J cleavage are nicked whereas synthetic hairpins are not. Cleavage-generated hairpins are nicked at the tip and predominantly 1 to 2 nt 5' of the tip. RAG1 and RAG2 may therefore be responsible for initiating the processing of coding ends and for the generation of P nucleotides during V(D)J recombination.     

Mutational analysis of terminal deoxynucleotidyltransferase-mediated N-nucleotide addition in V(D)J recombination

The addition of nontemplated (N) nucleotides to coding ends in V(D)J recombination is the result of the action of a unique DNA polymerase, TdT. Although N-nucleotide addition by TdT plays a critical role in the generation of a diverse repertoire of Ag receptor genes, the mechanism by which TdT acts remains unclear. We conducted a structure-function analysis of the murine TdT protein to determine the roles of individual structural motifs that have been implicated in protein-protein and protein-DNA interactions important for TdT function in vivo. This analysis demonstrates that the N-terminal portion of TdT, including the BRCA-1 C-terminal (BRCT) domain, is not required for TdT activity, although the BRCT domain clearly contributes quantitatively to N-nucleotide addition activity. The second helix-hairpin-helix domain of TdT, but not the first, is required for activity. Deletional analysis also suggested that the entire C-terminal region of TdT is necessary for N-nucleotide addition in vivo. The long isoform of TdT was found to reduce N-nucleotide addition by the short form of TdT, but did not increase nucleotide deletion from coding ends in either human or rodent nonlymphoid cells. We consider these results in light of the recently reported structure of the catalytic region of TdT.     

Modulation of RAG/DNA complex by HSP70 in V(D)J recombination

V(D)J recombination, a site-specific gene rearrangement process, requires two RAG1 and RAG2 proteins specifically recognizing recombination signal sequences and forming DNA double-strand breaks. The broken DNA ends tightly bound to RAG proteins are joined by repair proteins. Here, we found that heat shock protein 70 was associated with RAG2 following two-step affinity chromatography purification. It was also co-immunoprecipitated with RAG2 in pro-B cells. Purified HSP70 protein disrupted RAG/DNA complexes assembled in vitro and also inhibited the V(D)J cleavage (both nick and hairpin formation) in a dose-dependent manner. This HSP70 action required ATP energy. These data suggest that HSP70 might play a crucial role in disassembling RAG/DNA complexes stably formed during V(D)J recombination.     

Evidence for Ku70/Ku80 association with full-length RAG1

Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.     

Roles of the "dispensable" portions of RAG-1 and RAG-2 in V(D)J recombination

V(D)J recombination is initiated by introduction of site-specific double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catalyze V(D)J recombination, although less efficiently than their full-length counterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and products. We found that while truncated RAG proteins generate lower levels of recombination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates, termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the "dispensable" regions of the RAG proteins impairs proper processing of recombination intermediates. Furthermore, we found that removal of portions of the dispensable regions of RAG-1 and RAG-2 affects the efficiency of product formation without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in V(D)J recombination.     

Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products.

Ku, a heterodimer of 70- and 86-kDa subunits, serves as the DNA binding component of the DNA-dependent protein kinase (DNA-PK). Cells deficient for the 86-kDa subunit of Ku (Ku86-deficient cells) lack Ku DNA end-binding activity and are severely defective for formation of the standard V(D)J recombination products, i.e., signal and coding joints. It has been widely hypothesized that Ku is required for protection of broken DNA ends generated during V(D)J recombination. Here we report the first analysis of V(D)J recombination intermediates in a Ku-deficient cell line. We find that full-length, ligatable signal ends are abundant in these cells. These data show that Ku86 is not required for the protection or stabilization of signal ends, suggesting that other proteins may perform this function. The presence of high levels of signal ends in Ku-deficient cells prompted us to investigate whether these ends could participate in joining reactions. We show that nonstandard V(D)J recombination products (hybrid joints), which involve joining a signal end to a coding end, form with similar efficiencies in Ku-deficient and wild-type fibroblasts. These data support the surprising conclusion that Ku is not required for some types of V(D)J joining events. We propose a novel RAG-mediated joining mechanism, analogous to disintegration reactions performed by retroviral integrases, to explain how formation of hybrid joints can bypass the requirement for Ku and DNA-PK.     

Targeted transposition by the V(D)J recombinase

Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by--and specifically targeted to--hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected.     

Comparison of the two murine terminal [corrected] deoxynucleotidyltransferase terminal isoforms. A 20-amino acid insertion in the highly conserved carboxyl-terminal region modifies the thermosensitivity but not the catalytic activity

Terminal deoxynucleotidyltransferase (TdT) catalyzes the addition of nucleotides to 3'-hydroxyl ends of DNA strands in a template-independent manner and has been shown to add N-regions to gene segment junctions during V(D)J recombination. TdT is highly conserved in all vertebrate species, with a second isoform, characterized by a 20-amino acid insertion near the COOH-terminal end, described only in the mouse. The two murine isoforms differ in their subcellular localization, and the long isoform (TdTL) has previously been found to be unable to add N-regions. Using purified protein produced in a high level expression system in Escherichia coli, we were able to carry out detailed catalytic comparisons of these two TdT isoforms. We discovered that TdTL exhibits terminal transferase activity with kinetic parameters similar to those of the conserved TdT isoform (TdTS). We observed, however, that TdTL is inactivated at physiologic temperature but stable at lower temperatures. This thermal sensitivity of TdTL polymerase activity is not correlated with a significant change in the circular dichroism spectrum of the protein. Thus, the 20-amino acid insertion in TdTL does not affect the catalytic activity but modifies the thermosensitivity.