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Double-Strand Signal Sequence Breaks in V(D)J Recombination Are Blunt, 5'-Phosphorylated, RAG-Dependent, and Cell Cycle Regulated

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Double-Strand Signal Sequence Breaks in V(D)J Recombination Are Blunt, 5'-Phosphorylated, RAG-Dependent, and Cell Cycle Regulated Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Double-strand signal sequence breaks in V(D)Jrecombination are blunt, 5'-phosphorylated, RAG-dependent, and celIcycle regulated Mark Schlissel, Andrei Constantinescu, Terri Morrow, Mike Baxter, and Albert Peng Departments of Medicine and Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA Immunoglobulin and T-cell receptor genes are assembled during lymphocyte development by a novel, highly regulated series of gene rearrangement reactions known as V(D)I recombination. All rearranging loci are flanked by conserved heptamer-nonamer recombination signal sequences. Gene rearrangement results in the imprecise fusion of coding sequences and the precise fusion of signal sequences. DNA molecules with double-stranded breaks near signal sequences have been detected in cells undergoing V(D)I recombination of the TCR8 locus. We have devised a ligation-mediated PCR assay that detects broken-ended molecules in purified genomic DNA. Using this assay we found that DNA breaks occurring precisely at the signal sequence-coding sequence junction are a general feature of V(D)I recombination, appearing in association with each type of rearranging immunoglobulin gene segment. We show that a significant fraction of these broken ends are blunt and 5'-phosphorylated. In addition, detection of these broken-ended signal sequences is dependent on the activity of RAG-1 and RAG-2, and is restricted to the Go/G1 phase of the cell cycle. The pattern of broken-ended molecules detected in cells at various stages of development reflects the activity of the V(D)J recombinase at different loci during B- and T-cell development. [Key Words: V(D)I recombination; RAG-l, RAG-2~ DNA breaks; gene rearrangement; B- and T-cell development] Received August 25, 1993~ revised version accepted October 13, 1993. Immunoglobulin and T-cell receptor {TCRI genes are as- feres with heavy-chain allelic exclusion, supporting the sembled during B- and T-cell development by a series of suggestion that VL protein may help to mediate ordered site-specific recombination reactions known as V(D)!re- gene rearrangement in B cells (Kitamura et al. 1991; Ki- combination {for review, see Altet al. 1992}. Although tamura and Rajewsky 1992}. the reactants IV, D, and [ gene segmentsl and products Molecular biological approaches have led to the iden- {functional Ig~, IgK, and Igk, and TCRa, TCR[~, TCR-~, tification of several gene products required for V(D)! re- and TCR~ genes) have been characterized extensively, combination. RAG-1 and RAG-2 were cloned on the ba- details regarding the reaction mechanism and its modes sis of their ability to activate recombination of an im- of regulation remain elusive. munoglobulin-like reporter construct upon transfection The gene rearrangement program is ordered and occurs into a fibroblast cell line (Schatz et al. 1989; Oettinger et in a lineage-specific pattern during lymphoid develop- al. 1990}. RAG-1 and RAG-2 are coordinately expressed ment. i~-Heavy-chain gene rearrangement usually pre- in immature B and T cells, are not expressed in mature cedes K-light-chain gene rearrangement during B-cell de- lymphocytes, and are both required for V(D)I recombi- velopment, and TCRI3 gene rearrangement precedes ~x nation. Their precise roles in recombination remain un- gene rearrangement during T-cell development. Immu- clear, but homozygous disruption of either gene results noglobulin heavy-chain D-tO-/H rearrangement occurs in in mice incapable of carrying out V(D)J recombination all B and some T cells, TCR D-to-[B rearrangement oc- and completely lacking mature B and T cells (Mombaerts curs in T and some B cells, but V-to-D[ rearrangement is et al. 1992; Shinkai et al. 1992}. A similar, but less se- strictly lineage specific (Ferrier et al. 1990). Recent evi- vere, phenotype is associated with the routine scid mu- dence supports the notion that products of D-tO-/H and tation {Hendrickson et al. 1988~ Malynn et al. 1988). Ho- V-to-DIll gene rearrangement regulate B-cell develop- mozygous scid mice display ineffective V(D)[ recombi- ment. Targeted mutation of the membrane exon of the nation resulting in large deletions at the sites of gene prevents efficient K gene rearrangement and inter- attempted coding sequence joining. Interestingly, both 2520 GENES & DEVELOPMENT 7:2520-2532 91993 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/93 $5.00 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Signal sequence breaks in V(D)!recombination lymphoid and nonlymphoid cells from scid mice show are limited by the fact that the products of immunoglob- increased sensitivity to DNA damaging agents, leading ulin gene rearrangement affect cell division and cell life to the hypothesis that the scid gene product is not nec- span; therefore, the detection of rearranged immuno- essarily lymphoid specific (Biedermann et al. 1991; Hen- globulin genes reflects the targeting of the V(D)Jrecom- drickson et al. 1991). By use of a cotransfection approach, binase only indirectly. Furthermore, these assays cannot two additional genetic loci were recently identified that distinguish between ongoing and completed gene rear- show mutant phenotypes involving both DNA damage rangement in a mixture of developing cells. We argue repair and V(D)I recombination (Taccioli et al. 1993). that the presence of locus-specific, broken-ended RSS In contrast to the relative lack of detailed information DNA indicates V(D)J recombinase activity at that par- on the enzymatic components of the V(D)Jrecombinase, ticular locus. We have used this assay to characterize the much is known about the structure of rearranging loci effects of the scid mutation and an Ig~ transgene on the and the products of rearrangement (Tonegawa 1983). All targeting of the V(D)I recombinase during B-cell devel- immunoglobulin and TCR gene segments are flanked by opment. highly conserved heptamer and nonamer recombination signal sequences (RSSs). The heptamer and nonamer are Results separated by nonconserved spacer DNA of either 12 or 23 A linker-ligation assay for broken-ended RSS DNA (+-1) nucleotides, and only gene segments with flanking RSSs that have dissimilar spacer lengths recombine with We devised a technique, based on ligation-mediated PCR one another. Recombination of gene segments results in [LMPCR (Mueller and Wold 1989)], that detects blunt- two products--a coding joint and a signal joint. The cod- ended DNA molecules associated with any characterized ing joint is an imprecise fusion of the coding regions of V locus in purified genomic DNA (Fig. 1). A partially dou- and D, D and J, or V and ] gene segments. Nucleotides are ble-stranded oligonucleotide linker (BW linker) is ligated frequently deleted and sometimes added (N and P nucle- to purified genomic DNA by use of T4 DNA ligase. The otides) to these joints (Desiderio et al. 1984; Lafaille et al. blunt, nonphosphorylated, asymmetric structure of the 1989). The existence of P nucleotides, short palindromic linker is such that it can ligate only in a single orienta- repeats at the junctions of coding sequences, might be tion to blunt, 5'-phosphorylated genomic DNA. Broken explained by a hairpin DNA reaction intermediate (La- DNA in association with a particular locus is then de- faille et al. 1989; Lieber 1992). In contrast, signal joints tected by a pair of nested PCR assays with locus-specific are most often precise, head-to-head fusions of heptam- primers (labeled 1 and 2 in Fig. 1) and a primer identical ers without addition or deletion of nucleotides (Lewis et to the ligated strand of the linker (BW-1 or BW-1H), fol- al. 1985). The structure of the signal joint led various lowed by Southern blot hybridization. The same ligated workers to suggest that an early step in the reaction genomic DNA sample can be used to map double- must include the recognition and cleavage of DNA at the stranded DNA breaks in association with multiple loci heptamer-coding sequence junction. by altering the specific primers used in the PCR step of Roth, Gellert, and their colleagues recently described the assay. two novel DNA molecules that are probably reaction We used this LMPCR technique to test DNA from intermediates in V(D)J recombination (Roth et al. various mouse tissues and cell lines for broken-ended 1992a, b). By use of Southern blot hybridization, these RSSs associated with various immunoglobulin gene loci workers detected double-stranded breaks associated with (Fig. 2). Linker-ligated DNA samples from newborn thy- RSSs and covalently closed DNA hairpins associated mus, 2-month-old wild-type bone marrow, 2-month-old with coding regions of the TCR~ locus in DNA purified scid bone marrow, and the Abelson virus-transformed from thymocytes undergoing TCR8 gene rearrangement. RAG-2-deficient cell line 63-12 [kindly provided by Dr. Interestingly, the hairpin ends could be detected only in Fred Alt (Shinkai et al. 1992)] were analyzed by locus- scid thymocytes. They proposed that these broken DNA specific PCR and Southern blot assays. Hybridizing frag- molecules were reaction intermediates in V(D)J recom- ments corresponding in size to breaks at various Jn, JK, bination with structures that might account for the ob- and Dfll6.1 RSSs were detected in certain tissues (ar- served products of the reaction as suggested above. rows). Control PCR assays of an unrelated genetic locus In this paper we describe a sensitive PCR-based assay (the CD14 gene) indicated that similar amounts of li- for locus-specific broken-ended DNA. We show that pre- gated DNA were used in each assay (lanes 16-20). cise broken-ended RSSs are associated with each rear- Dill6.1 is the most JR-distal of the known mouse DR ranging immunoglobulin locus in cells undergoing V(D)J genes and was shown to be involved in almost 50% of recombination.
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