The composition of coding joints formed in V(D)J recombination is strongly affected by the nucleotide sequence of the coding ends and their relationship to the recombination signal sequences.

V(D)J recombination proceeds in two stages. Precise cleavage at the border of the conserved recombination signal sequences (RSSs) and the coding ends results in flush double-stranded signal ends and coding ends terminating in hairpins. In the second stage, the signal and coding ends are processed into signal and coding joints. Coding ends containing certain nucleotide homopolymers affect the efficiency of V(D)J recombination. In this study, we have tested the effect of small changes in coding-end nucleotide composition on the frequency of coding- and signal joint formation. Furthermore, we have determined the sequences of coding joints resulting from recombination of coding ends with different compositions. We found that the presence of two T nucleotides 5' of both RSSs, but not a single T, reduces the frequency of signal joint formation, i.e., interferes with the cleavage stage of V(D)J recombination. However, coding-joint processing is sensitive even to a single T. Both the sequence of the coding ends and the particular RSS (12-mer or 23-mer) with which the coding end is associated affect the final composition of the coding joints. Thus, the presence of P nucleotides, the conservation of one undeleted coding end, the formation of joints without any deletions, and the template-dependent insertion of nucleotides are strongly influenced by the coding-end nucleotide composition and/or RSS association. The implications of these results with respect to the processing of coding ends are discussed.     

Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

The V(D)J recombinase recognizes a pair of immunoglobulin or T-cell receptor gene segments flanked by recombination signal sequences and introduces double-strand breaks, generating two signal ends and two coding ends. Broken coding ends were initially identified as covalently closed hairpin DNA molecules. Before recombination, however, the hairpins must be opened and the ends must be modified by nuclease digestion and N-region addition. We have now analyzed nonhairpin coding ends associated with various immunoglobulin gene segments in cells undergoing V(D)J recombination. We found that these broken DNA ends have different nonrandom 5′-strand deletions which were characteristic for each locus examined. These deletions correlate well with the sequence characteristics of coding joints involving these gene segments. In addition, unlike broken signal ends, these nonhairpin coding-end V(D)J recombination reaction intermediates have 3′ overhanging ends. We discuss the implications of these results for models of how sequence modifications occur during coding-joint formation.     

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.     

V(D)J recombination coding junction formation without DNA homology: processing of coding termini.

Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining.     

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.     

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.     

Sequence and hairpin structure of an inverted repeat series at termini of the Physarum extrachromosomal rDNA molecule

The termini of the 61 kb palindromic rDNA molecules of Physarum polycephalum possess a series of multiple inverted repeats in which are located specific single-strand gaps and tightly attached protein. After treating rDNA with S1 nuclease, we have cloned several 5 kb Eco RI terminal restriction fragments. Sequencing of more than 800 nucleotides from the end of one such clone reveals the presence of six to ten tandemly repeated units averaging 140 +/- 4 bp in length and flanked by Hae III sites. Each 140 nucleotide repeat unit can form thermodynamically stable hairpin structures based on complex internal palindromic components. When the specific gap sequence CCCTA is present, it is located near the apex of a hairpin component. These secondary structures are formed in growing plasmodia, as seen in electron micrographs of native rDNA molecules, which also reveal apparent recombination forms involving rDNA ends and noncontiguous DNA segments. Recombination initiated at terminal single-strand hairpin loops can result in genetic exchange of ribosomal gene sequences and can lead to completion of 5' nucleotide sequences at ends of newly replicated rDNA molecules.     

A transposon-related palindromic repetitive sequence from C. elegans.

A family of transposon-like sequences in the C. elegans genome is described. This family, termed the Tc6 family, consists mostly of conserved, 1.6 kb elements. Four Tc6 elements or partial elements have been cloned and the DNA sequences of three were determined. One appears to be a complete element of 1603 nucleotides, consisting of a palindrome of 765 nucleotides, with a central, non-palindromic region of 73 nucleotides. Another has an identical structure except for an internal deletion. A third is a partial element terminating at a probable internal restriction site used for cloning. A fourth clone contained portions of the Tc6 sequence juxtaposed to non-Tc6 sequences. All C. elegans strains examined contain 20-30 Tc6 elements. The ends of Tc6 elements are conserved and have sequence similarity to the ends of C. elegans transposons Tc1 and Tc3. The ends of Tc6 elements also have sequence similarity to the heptamer portion of the immunoglobulin and T-cell receptor recombination signal sequence, raising the possibility of wide phylogenetic conservation of the recombination mechanism. Tc6 elements also share sequence motifs with plant-pathogenic viroid RNA's, possibly indicative of a Tc6 RNA replicative phase.     

Possible role of nucleotide correlations between human exon junctions

There is ample evidence that prediction of human splice sites can be refined by analyzing the nucleotides surrounding splice sites. This could mean that exon nucleotides over splice sites harbour information for the splicing process in addition to the coding information to specify aminoacids. We analyzed the correlations among the nucleotides lying at the end and at the beginning of all the consecutive human exons to seek relationships among the nucleotides. We have divided the sequences taking into account the phase of interruption. Even though exon sequences are involved in the coding function, we found phase-dependent, specific correlations in the area of exon junctions. These regularities do not give rise to specific motifs, but rather to a phase-specific nucleotide context that could contribute to define the splice site or aid the splicing machinery to join the exon ends. Results provide further evidence that accurate selection of human splice sites likely requires the contribution of exon regulatory sequences.     

Variable diversity joining recombination: nonhairpin coding ends in thymocytes of SCID and wild-type mice

Initiation of V(D)J recombination results in broken DNA molecules with blunt recombination signal ends and covalently sealed (hairpin) coding ends. In SCID mice, coding joint formation is severely impaired and hairpin coding ends accumulate as a result of a deficiency in the catalytic subunit of DNA-dependent protein kinase, an enzyme involved in the repair of DNA double-strand breaks. In this study, we report that not all SCID coding ends are hairpinned. We have detected open Jdelta1 and Ddelta2 coding ends at the TCRdelta locus in SCID thymocytes. Approximately 25% of 5'Ddelta2 coding ends were found to be open. Large deletions and abnormally long P nucleotide additions typical of SCID Ddelta2-Jdelta1 coding joints were not observed. Most Jdelta1 and Ddelta2 coding ends exhibited 3' overhangs, but at least 20% had unique 5' overhangs not previously detected in vivo. We suggest that the SCID DNA-dependent protein kinase deficiency not only reduces the efficiency of hairpin opening, but also may affect the specificity of hairpin nicking, as well as the efficiency of joining open coding ends.     

Mechanistic basis for coding end sequence effects in the initiation of V(D)J recombination

V(D)J recombination is directed by recombination signal sequences. However, the flanking coding end sequence can markedly affect the frequency of the initiation of V(D)J recombination in vivo. Here we demonstrate that the coding end sequence effect can be qualitatively and quantitatively recapitulated in vitro with purified RAG proteins. We find that coding end sequence specifically affects the nicking step, which is the first biochemical step in RAG-mediated cleavage. The subsequent hairpin formation step is not affected by the coding end sequence. Furthermore, the coding end sequence effect can be ablated by prenicking the substrate, indicating that the coding end effect is specific to the nicking step. In reactions in which both 12- and 23-substrates are present, a suboptimal coding end sequence on one signal can slow down hairpin formation at the partner signal, a result consistent with models in which coordination between the signals occurs at the hairpin formation step. The coding end sequence effect on nicking and the coupling of the 12- and 23-substrates explains how hairpin formation can be rate limiting for some 12/23 pairs, whereas nicking can be rate limiting when low-efficiency coding end sequences are involved.     

The presence of direct repeats does not influence coding joint formation during V(D)J recombination.

During the recombination process that assembles immunoglobulin and T-cell receptor gene segments, the coding ends to be joined are extensively processed. Contradictory reports have been made in the past about the existence of homology directed mechanisms in V(D)J recombination. In this study we analyse coding end processing and the influence of the presence of homology stretches on coding joint formation using artificial substrates in which short sequence changes creating direct repeats have been introduced. These changes were monitored 3 bp away from the termini in order to avoid any differences due to the initiation steps of V(D)J recombination. Our results show that the sequence of the coding ends influences joint formation, but no evidence was found for a mechanistic bias due to the presence of direct repeats.     

V(D)J recombinase-mediated processing of coding junctions at cryptic recombination signal sequences in peripheral T cells during human development

V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (Pr-nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age- and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans.     

Resolution of poxvirus telomeres: processing of vaccinia virus concatemer junctions by conservative strand exchange.

The replication of vaccinia virus proceeds through concatemeric intermediates which are resolved into unit-length DNA. In vaccinia virus-infected cells, plasmids containing the vaccinia virus DNA junction fragment that connects concatemers are resolved into linear minichromosomes of vector DNA flanked by hairpin loops. Resolution requires two copies of a specific nucleotide sequence conserved among poxviruses and found proximal to the hairpin loop. This study demonstrates that orientation of each sequence with respect to the other as well as to the axis of symmetry is critical for resolution, the processing of plasmids containing heterologous pairs of resolution sites is influenced by mismatched nucleotides between the sites, and the vaccinia virus hairpin in the linear minichromosome is a heteroduplex composed of DNA from each strand of the concatemer junction. A model incorporating site-specific recombination and orientated branch migration is proposed to account for resolution of the vaccinia virus concatemer junction.     

Hairpin opening by single-strand-specific nucleases.

DNA molecules with covalently sealed (hairpin) ends are probable intermediates in V(D)J recombination. According to current models hairpin ends are opened to produce short single-stranded extensions that are thought to be precursors of a particular type of extra nucleotides, termed P nucleotides, which are frequently present at recombination junctions. Nothing is known about the activities responsible for hairpin opening. We have used two single-strand-specific nucleases to explore the effects of loop sequence on the hairpin opening reaction. Here we show that a variety of hairpin ends are opened by P1 nuclease and mung bean nuclease (MBN) to leave short, 1-2 nt single-stranded extensions. Analysis of 22 different hairpin sequences demonstrates that the terminal 4 nt of the hairpin loop strongly influence the sites of cleavage. Correlation of the nuclease digestion patterns with structural (NMR) data for some of the hairpin loops studied here provides new insights into the structural features recognized by these enzymes.     

Characterization of Wild-Type Adeno-Associated Virus Type 2-Like Particles Generated during Recombinant Viral Vector Production and Strategies for Their Elimination

The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.     

Joining-deficient RAG1 mutants block V(D)J recombination in vivo and hairpin opening in vitro

The RAG proteins cleave at V(D)J recombination signal sequences then form a postcleavage complex with the broken ends. The role of this complex in end processing and joining, if any, is undefined. We have identified two RAG1 mutants proficient for DNA cleavage but severely defective for coding and signal joint formation, providing direct evidence that RAG1 is critical for joining in vivo and strongly suggesting that the postcleavage complex is important in end joining. We have also identified a RAG1 mutant that is severely defective for both hairpin opening in vitro and coding joint formation in vivo. These data suggest that the hairpin opening activity of the RAG proteins plays an important physiological role in V(D)J recombination.     

Sequence of human adenosine deaminase cDNA including the coding region and a small intron

The nucleotide sequence for an unusual, cloned human adenosine deaminase cDNA has been determined. Contained within a sequence of 1535 nucleotides is a coding sequence of 1089 nucleotides that encodes a protein of 40,762 daltons. The coding sequence is interrupted by a non-coding region containing 76 nucleotides. Both the 3' and 5' ends of this region have consensus sequences generally associated with splice sites. The 3' untranslated sequence contained 308 nucleotides, including a polyadenylation signal sequence 20 nucleotides from the end. The cloned cDNA appears to correspond to a nuclear mRNA precursor which contains a small intron.     

V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes.

Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor.     

Failure of Hairpin-Ended and Nicked DNA To Activate DNA-Dependent Protein Kinase: Implications for V(D)J Recombination

V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PK_CS or p460). Ku directs DNA-PK_CS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PK_CS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PK_CS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.