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

Cell, Vol. 70, 983-991,

September

19, 1992, Copyright

0 1992 by Cell Press

V(D)J Recombination: Broken DNA Molecules with Covalently Sealed (Hairpin) Coding Ends in scid Mouse Thymocytes David B. Roth,* Joseph P. Menetski,* Pamela 9. Nakajima,t Melvin J. Bosma,t and Martin Gellert’ *Laboratory of Molecular Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892 tlnstitute for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 19111

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 TCRG 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.

CUleS with coding ends, suggesting that coding ends might be joined (or degraded) more rapidly than signal ends. Since the autosomal recessive mouse mutation scid is thought to impair specifically the joining of coding ends (Lieber et al., 1988; Blackwell et al., 1989) we wondered whether molecules with coding ends might accumulate in mice homozygous for this mutation (scid mice). scid maps to chromosome 16 and results in an apparent arrest of early T and B cell development. Cells committed to the T lineage are present in the thymus of scid mice, but functional T cells (TCR’) are absent (reviewed by Bosma and Carroll, 1991). Recent work indicates that the TCRG locus is recombinationally active in both newborn and adult scid thymocytes (Carroll and Bosma, 1989, 1991). We therefore examined DNA preparations from scid thymocytes for broken DNA molecules. We report here the presence of nongermline restriction fragments in scid thymus DNA of sizes appropriate to terminate in both signal ends and coding ends. We demonstrate that fragments with coding ends, but not signal ends, have a uniquely altered mobility in twodimensional electrophoresis’that is explained by the presence of a covalent bond between the two strands of the coding end, forming a hairpin. These findings support a refined model for V(D)J recombination that incorporates the formation of hairpins at coding ends as a possible early step and suggest that the scid factor may be involved in the resolution of hairpins.

Results Introduction V(D)J recombination is the process that assembles complete immunoglobulin and T cell receptor (TCR) variable regions from separate germline coding segments. V(D)J assembly occurs in developing lymphoid cells and relies on recognition of the specific signal sequences adjacent to the coding segments. Recombination generates two types of junctions, coding joints (the junction between coding segments) and signal joints (the reciprocal junction consisting of a precise fusion of the recombination signals) (Lewis and Gellert, 1989). One possible mechanism for this reaction is diagrammed in Figure 1. According to this scheme, double-strand breaks are made at the borders between the signal sequences and the coding segments, generating molecules with two types of termini, termed coding ends and signal ends. Subsequent joining of the coding ends fuses the gene segments, producing a coding joint. Joining of the signal ends forms the reciprocal product, in this case a circular molecule containing a signal joint. This model is supported by the recent identification of DNA molecules with double-strand breaks near TCRG recombination signal sequences; excised linear molecules with signal ends, as well as circular reciprocal products containing signal joints, were observed in thymocyte DNA (Roth et al., 1992). Although molecules with signal ends were relatively abundant, we could not detect mole-

Broken DNA Molecules with Coding Ends in scid Thymocytes The D2 and Jl elements of the TCRS locus participate in a large fraction of the rearrangements observed in newborn and young adult mice (Chien et al., 1987; Elliott et al., 1988). Cleavage events involving these elements could generate a number of possible products, as shown in Figure 2. Previously, only molecules with signal ends (the 2500 bp left D signal and the 900 bp excised linear fragment shown in Figure 2) were detected in thymocytes from wild-type (BALBlc) mice (Roth et al., 1992). To determine whether the scid mutation results in the accumulation of molecules with coding ends, thymus DNA preparations from adult scid mice were digested with EcoRl and analyzed by Southern blotting. Double-strand breaks at the recombination signal sequences flanking J&l or Ds2 would generate fragments of 4000 bp and 4900 bp, respectively, that should hybridize to the 3’Jsl probe (Figure 2). As shown in Figure 3A, DNA from adult scid thymus (lane 2) contains an abundant germline fragment (7400 bp); in addition, as compared with wild type (lane l), two fragments of 4900 bp and 4000 bp are prominent. (Since these species are not seen in undigested DNA preparations, they are not excised linear or circular molecules.) In wild-type thymus DNA there are other fragments that presumably correspond to completed rearrangements, in-

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1. A Breakage-Reunion

Jl

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for V(D)J Recombination

b-4 900 bp

Coding segments are represented by rectangles. Recombination signal sequences are shown as triangles, with heptamers adjacent to the coding segments. Signals with 23 and 12 nt spacers are designated by closed and open triangles, respectively.

eluding a 6600 bp fragment resulting from D2-Jl joining (see below). Digestion of scid thymus DNA with BamHI, which cleaves approximately 200 bp to the right of the EcoRl site (see Figure 2) produces fragments of about 4200 bp and 5100 bp (data not shown). The observation that the fragment sizes change predictably upon digestion with BamHl suggests that the 4000 bp and 4900 bp EcoRl species are broken molecules rather than completed rearrangements. This conclusion is supported by additional data described below. Rehybridization of the membrane shown in Figure 3A to a probe specific for sequences between D2 and Jl (DJ probe; see Figure 2) demonstrated that the 4900 bp fragment, but not the 4000 bp fragment, hybridizes to the DJ probe (Figure 38). This behavior is expected for molecules resulting from double-strand breaks near D62 and Jsl , respectively. Because J&l is flanked by a single recombination signal (on the left side), cleavage is expected to generate a 4000 bp species that terminates in a coding end (diagrammed in Figure 2). Additional experiments described below indicate that both the 4000 bp and 4900 bp fragments terminate in coding ends. EcoRl digestion of newborn BALBlc thymus DNA (Figure 3A, lane 1) generates several fragments, including germline (7400 bp), and several other species that are thought to result from completed rearrangements (Roth et al., 1992); however, no 4900 bp or 4000 bp fragments are apparent. Similar results are obtained with DNA from fetal or young adult (5-6 weeks) BALBlc thymocytes. In a few experiments, a faint band corresponding to the 4000 bp fragment is visible (data not shown). Quantitation of this weak signal is difficult, but we estimate that this fragment is less than 10% as abundant in BALBlc thymus DNA as in scid thymus DNA. No fragment of 3400 bp, which would correspond to molecules with signal ends cleaved at Jl (see Figure 2) is apparent (Figure 38) indicating that molecules cleaved at only a single signal (at the Jl signal but not the partner signal at D2) do not accumulate in scid

Right D Signal 4900 bp

Coding Ends

J Coding 4000 bp

Right D Codtng

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Figure 2. Possible Broken Molecules Resulting from Double-Strand Cleavage at TCRG D2 and Jl Recombination Signals The closed rectangles above the restriction map represent the DNA sequences used as hybridization probes. The coding elements and recombination signal sequences are not drawn to scale. Potential cleaved species are shown as EcoRl digestion products; the expected lengths of the resulting restriction fragments are shown. Restriction sites are designated as follows: B, BamHI; E, EcoRI; X, Xmnl (see Roth et al., 1992, for a more detailed restriction map of the TCRG locus). Symbols are as in Figure 1.

thymocytes, in agreement with previous results from wildtype thymocytes (Roth et al., 1992). Hairpin Coding Ends in scid Thymocytes Why does the scid mutation result in the accumulation of molecules with coding ends? Possibly there is less joining activity, leading to an increase in the lifetime of broken molecules. Alternatively, the scid defect may block the processing of coding ends at an early stage, leading to their accumulation in a modified form that is resistant to joining (or perhaps to degradation), which might explain their abundance. To test for end modification, we challenged DNA preparations from scid thymocytes with Mi-

Hairpin 985

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3. Detection

of Putative

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in scid Thymocytes

(A) Genomic DNA (8 kg) was digested with EcoRI. and the fragments were separated by electrophoresis through a 1% agarose gel. The membrane was hybridized to the 3’Jsl probe. The sizes of the relevant fragments are indicated. (B) The membrane shown in (A) was stripped and rehybridized to the DJ probe. The 3400 bp position, where a fragment resulting from cleavage only at the recombination signal sequence adjacent to Ji would appear, is indicated by an arrowhead.

crococcus luteus ATP-dependent exonuclease. Although we previously found molecules with signal ends isolated from wild-type thymocytes to be completely sensitive to exonuclease (Roth et al., 1992), both the 4000 bp and 4900 bp fragments from scid thymocytes are exonuclease resistant (data not shown), suggesting that the ends are modified in some way. Certain features of coding joint sequences have led to the suggestion that hairpin ends might be intermediates in the normal pathway of V(D)J recombination (see Discussion). We therefore considered the possibility that the coding ends in scid thymocytes might contain a covalent bond between the terminal bases of the two strands, creating a hairpin. To explore the structural characteristics of the ends in more detail, two-dimensional native/alkaline gel electrophoresis was performed. EcoRI-digested molecules were separated first under neutral conditions, followed by electrophoresis through an alkaline agarose gel. In this system, molecules with unprocessed ends (that is, blunt or nearly blunt double-stranded termini) should migrate with

similar mobilities in both dimensions, forming a diagonal. The apparent sizes of molecules with hairpin ends will double in the alkaline dimension, causing these species to migrate above the diagonal. Analysis of DNA from scid thymocytes (Figure 4A) using the 3’Jsl probe revealed two species above the diagonal, with mobilities in the native dimension of 4000 bp and 4900 bp. In the alkaline dimension, the apparent sizes of these fragments double, as predicted for molecules with covalently sealed ends. Rehybridization of the membrane to the DJ probe revealed the 4900 bp, but not the 4000 bp, species (Figure 48) confirming the results obtained with native gel electrophoresis (see Figure 3). Two-dimensional gel analysis of thymus DNA from wild-type mice using the 3’Jal probe (data not shown) or the 5’Da2 probe (see below) failed to demonstrate molecules with hairpin ends. These data indicate that molecules containing doublestrand breaks near D2 and Jl are present in scid thymocytes and that their termini are covalently sealed. This conclusion is supported by the results of Sl nuclease experiments. Treatment with Sl, which nicks hairpin structures (Lilley, 1980; Panayotatos and Wells, 1981) dramatically reduces the intensity of the 4000 bp and 4900 bp species migrating above the diagonal (Figure 4C). As described above, the presence of a single recombination signal sequence to the left of J&l leads one to expect that the 4000 bp fragment generated by digestion with EcoRl terminates in a coding end. However, since recombination signal sequences are present on both sides of D2, the 4900 bp fragment could correspond to molecules with either right D coding ends or right D signal ends (see Figure 2). These fragments would differ by only 16 nt (the length of the D element) and would not be resolved by the electrophoresis conditions used here. The observation that the majority of the 4900 bp fragments terminate in hairpins suggests that most of these fragments have coding ends; however, the presence of some molecules with right D signal ends cannot be ruled out. Since the 4900 bp fragment has not been detected in wild-type thymocytes (see Figure 3), in contrast with other fragments with known signal ends (Roth et al., 1992), it seems unlikely that molecules with signal ends comprise a significant fraction of the 4900 bp species. Our inability to detect such fragments (predicted to be associated with D2-J2 joining events) may reflect the low frequency of D2-J2 rearrangements in thymus DNA (Chien et al., 1987; Elliott et al., 1988; D. B. R., J. P. M., P. B. N., M. J. B., and M. G., unpublished data). Absence of Hairpins at Signal Ends The .5’Da2 probe was used to search for other species resulting from cleavage near Ds2 (Figure 5). Several fragments are present in EcoRI-digested DNA from BALBlc thymocytes (Figure 5A, lane l), including germline (7400 bp), a 6600 bp fragment resulting from D2-Jl rearrangement (Chien et al., 1987; Carroll and Bosma, 1991), several species with mobilities between 3300 bp and 3000 bp, and a prominent 2500 bp fragment. Our previous work has shown that in wild-type cells, the 2500 bp species results from a double-strand break at Ds2 and terminates in a

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Frgure 4. Two-Dimensional Native/Alkaline Gel Electrophoresis Demonstrates the Presenceof Hairpin Termini

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signal end (Roth et al., 1992). The fragments migrating between 3000 bp and 3300 bp are generated by EcoRl digestion of circular excision products of Dal-D62 and V-D62 rearrangements; these molecules contain signal joints (Roth et al., 1992; D. B. R., J. P. M., P. B. N., M. J. B., and M. G., unpublished data). When subjected to two-dimensional native/alkaline gel electrophoresis, all of these fragments migrate on the diagonal (Figure 58). No 2500 bp fragments can be seen above the diagonal, even on very long autoradiographic exposures. Therefore, this signal end does not accumulate as a hairpin structure in wild-type thymocytes. This finding is consistent with our earlier observation that fragments containing signal ends are completely exonuclease sensitive (Roth et al., 1992). DNA from adult scid thymus presents a different picture (Figure 5A, lane 2), with a prominent germline fragment and very little of the 6600 bp fragment resulting from D2-Jl rearrangement. This reflects the low frequency of normal rearrangements observed in TCR loci of scid mice (Schuler et al., 1986,1988; Carroll and Bosma, 1991). Additional fragments of approximately 5500 bp and 3000 bp are present, as well as the 2500 bp species that presumably results from double-strand breaks near D&2. Upon inspection of the autoradiogram in Figure 5A, the abundance of the 2500 bp fragment in scid and BALBlc thymocytes appears similar; in wild-type thymocytes, this fragment represents approximately 2% of total genomic DNA (Roth et al., 1992). These results have been confirmed by quantitative autoradiography. More detailed analysis revealed a major difference between DNA preparations from scid and wild-type thymocytes. In two-dimensional gel electrophoresis of scid thymus DNA, one sees that approximately 50% of the molecules migrating at 2500 bp under native conditions migrate at twice this size under denaturing conditions (Figure 5C), demonstrating the presence of hairpin termini.

C

(A) Adult scid thymus DNA (7 ug) was digested with EcoRI; the fragments were separated by two-dimensional gel electrophoresis as described in Experrmental Procedures. The membrane was hybridized to the 3’Jhl probe. The direchons of migration in the native and denaturing (alkaline) dimensions are designated by arrows labeled N and D, respectively. The sizes of the relevant fragments (in the native dimension) are indicated. (B) The membrane shown in (A) was stripped and rehybridized to the DJ probe. Only the relevant portion of the autoradiogram is shown rn this enlarged view. For comparison, the same region of the autoradiogram from (A) is shown below at the same magnification. (C) Adult scid thymus DNA (7 ug) was treated with Sl nuclease, digested with EcoRI, and subjected to two-dimensional gel electrophoresis. The membrane was hybridized to the 3’J,l probe

The portion of the 2500 bp species migrating on the diagonal was completely sensitive to exonuclease, whereas the portion migrating above the diagonal was resistant to exonuclease, as expected (data not shown). Since the Ds2 element participates in both leftward (V to D2 or Dl to D2) and rightward (D2 to J) rearrangements, double-strand breaks might occur at either of the recombination signal sequences flanking 02, generating species terminating in D2 signal or D2 coding ends (see Figure 2). The presence of two distinct populations of molecules with different end structures suggests that both D2 coding (hairpin) and D2 signal (nonhairpin) ends might be present in scid thymocytes. We performed additional restriction mapping of these species to test this possibility. High Resolution Mapping of Coding and Signal Ends To distinguish between molecules with 02 signal and D2 coding ends, which differ in length by only 16 nt, DNA preparations were digested with Xmnl, which cleaves 298 bp to the left of D&2, and separated by electrophoresis through a NuSieve agarose gel. We previously used this strategy (diagrammed in Figure 6A) to map D2 signal ends in BALBlc thymocyte DNA (Roth et al., 1992). Size standards were constructed by digestion of p5’Da2 plasmid DNA with Xmnl and either Dpnl, which cleaves near the left border of the D62 element (299 bp fragment), HgiAI, which cuts immediately to the right of Da2 (314 bp fragment), or Pstl, which cleaves 40 bases to the right of Ds2 (357 bp fragment). As shown in Figure 6B, Xmnl digestion of scid thymus DNA produces two discrete fragments that are present in roughly equivalent amounts (Figure 6B, lanes 3-4). The smaller fragment migrates close to the position of the Dpnl-Xmnl marker, as expected for a signal end, and the larger fragment migrates close to the position of the HgiAl-Xmnl marker, as expected for a coding end.

Hairpin 907

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Ends

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Figure 5. Analysis of Molecules Cleaved f&2 from scid and Wild-Type Thymocytes

(A) Genomic DNA (8 pg) was digested with EcoRI, and the fragments were separated by electrophoresis through a 1% agarose gel. The sizes of the relevant fragments are indicated. The origin of the fragment migrating at approximately 5300 bp in scid thymus DNA has not been determined. (6) Newborn BALBlc thymus DNA (25 pg) was digested with EcoRI, and the fragments were separated by two-dimensional gel electrophoresis. (C)Adult scid thymus DNA (7 Fg) was digested with EcoRI, and the fragments were separated by two-dimensional gel electrophoresis. The faint doublet migrating above the diagonal at approximately 7400 bp is not reproducible. All three membranes were hybridized to the 5’Ds2 probe.

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scid

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Only the smaller fragment, corresponding to a signal end, is visualized in wild-type thymocytes (Figure 68, lanes 5-6). To test for the presence of hairpin structures at either the coding or the signal ends, undigested thymus DNA was treated with exonuclease, followed by digestion with Xmnl. As shown in Figure 6B (lanes 7-6) in scid thymus DNA only the smaller fragment, corresponding to molecules with signal ends, is susceptible to exonuclease. This fragment is also sensitive to exonuclease in BALBlc thymus DNA (Figure 6B, lanes 11-12) in agreement with previous results (Roth et al., 1992). Although there are many factors that might cause a broken end to be exonuclease insensitive, the presence of diagnostic species on two-dimensional gels (see Figure 5C) indicates that in this case the resistance of these coding ends to exonuclease is due to the presence of hairpin structures. Excised Linear Fragments and Circular Reciprocal Products in scid Thymocytes In DNA preparations from wild-type thymocytes, we have detected excised linear molecules with signal ends at both termini resulting from double-strand cleavage at the recombination signal sequences flanking D62 and J&l (Roth et al., 1992). The corresponding circular molecules, which contain signal junctions, were also detected. The linear molecule is 900 bp in length (see Figure 2); the circular form migrates with an apparent mobility of 700 bp. These species are present in both EcoRI-digested and undigested DNA preparations from scid (Figure 7, lanes 3-4) as well as wild-type (lanes l-2) thymocytes. The ratio of linear to circular species is somewhat increased in scid thymocytes, which might reflect either a subtle feature of the scid recombination defect or differences in thymocyte populations. The excised linear fragments from both scid and wild-type thymocytes are sensitive to digestion with exonuclease (data not shown), as expected for molecules with signal ends; the circular species is resistant to exonuclease. The presence of excised linear molecules and circular reciprocal products is consistent with previous

at

studies using extrachromosomal recombination substrates transfected into scid cell lines, which demonstrated that the initiationof recombination, as well assignal jointformation, is not greatly impaired by the scici defect (Lieber et al., 1988). Discussion A large body of evidence indicates that the murine scid mutation impairs the process of antigen receptor gene rearrangement in developing B and T cell precursors by interfering specifically with the formation of coding joints (Lieber et al., 1988; Blackwell et al., 1989); however, the biochemical step at which thescidgene product acts is not known. The data presented here demonstrate that broken DNA molecules with coding ends are relatively abundant in thymocytes from scid mice. These species are formed by cleavage near the D2 and Jl elements of the TCRG locus and are at least 1O-fold more abundant in scid than in wild-type thymocyte DNA. Surprisingly, coding ends isolated from scid thymocytes have a unique structure: the majority are sealed into hairpins. We have identified three species with coding ends, as well as two species with signal ends, in thymocytes from scid mice, as summarized in Figure 8. Of the molecular species with hairpin termini, one is broken immediately to the right of the D&2 element. This fragment terminates in a coding end, as shown by high resolution DNA blotting analysis, which maps the terminus to within 5 nt of the right end of D2. No heterogeneity in the length of these molecules is apparent, suggesting that cleavage occurs at a unique site. Molecules with covalently sealed coding ends resulting from cleavage near J&l are also seen in scid thymocytes (the 4000 bp EcoRl fragment). The 4900 bp fragment visualized with the 3’Jal and DJ probes also terminates in a hairpin end and most probably represents a coding end generated by cleavage to the left of D2. Electrophoresis under denaturing conditions results in a 2-fold increase in the apparent sizes of the 2500, 4000,

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Ftgure 7. Excised Linear Fragmentsand of D2-Jl Rearrangement

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. Figure 6. Mapping the Position Digestion with Xmnl

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(A) The nucleotide sequence of the D2 element is shown. The heptamer elements of the recombination signal sequences are boxed; expected sites of cleavage are marked by open arrows. Positions of cleavage by restriction enzymes are shown as closed arrows. The restriction fragments used as size markers and the expected products of double-strand breaks on either side of D2 are diagrammed. Symbols are as in Figure 1. The length of the Xmnl-HgiAl fragment was calculated from the Xmnl site to the center of the HgiAl site. (B) DNA fragments were resolved by electrophoresis through a 3% NuSieve agarose gel. The blot was hybridized to the 5’Da2 probe. Markers were produced by digesting a gel-purified Xmnl-Pstl fragment, obtained by digestion of the p5’Dn2 plasmid with either Dpnl or HgiAI. Lane 1 contains 0.3 pg of an equal mixture of Xmnl-Dpnl (marking the left end of D2) and Xmnl-Pstl fragments. Lane 2 contains 0.1 pg of the Xmnl-HgiAl fragment that marks the right end of D2. Lanes 3-6 contain the indicated amounts of Xmnl-digested thymus DNA. For lanes 7-8, the indicated amounts of scid thymus DNAwere treated with exonuclease priortodigestion withxmnl, asdescribed in Experimental Procedures. (The actual amounts of DNA loaded in these lanes and in lanes 11-12 may be less because of variable losses during extraction after treatment with exonuclease.) Lanes 9 and IO were loaded identically to lanes 2 and I, respectively. For lanes 1 l-l 2, the indicated amounts of newborn BALBlc thymus DNA were treated with exonuclease prior to digestion with Xmnl. Lanes 13-15 contain 0.1 pg of the Xmnl-HgiAl marker fragment, mixed with the indicated amounts of Xmnl-digested BALBlc liver DNA. These lanes demonstrate that the relative mobility of this fragment is not affected by differences in the amount of Xmnl-digested genomic DNA loaded on the gel.

Circular

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Genomic DNA (9 pg) was subjected to electrophoresis on a 1% agarose gel in the absence of ethidium bromide. The membrane was hybridized to the DJ probe. The apparent mobilities of the circular and linear forms, as determined by comparison with ethidium bromidestained size markers, are indicated. (These species are not seen in the blot shown in Figure 38, as fragments less than 1 kb were not retained on the gel.)

and 4900 bp fragments, indicating that the coding termini are covalently sealed. We have obtained no evidence for other forms of processing, such as the creation of long single-strand tails through the action of exonucleases, as seen at double-strand breaks formed during homologous recombination in yeast (White and Haber, 1990; Sun et al., 1991).

Hairpins

and the Origin of P Nucleotides

One curious feature sometimes found at coding joints is the insertion of a few extra nucleotides that are complementary to the coding end to which they are attached (the so-called P nucleotides) (Lafaille et al., 1989; McCormack et al., 1989). It was initially suggested that one strand of a coding end might be nicked 2 nt from the end, liberating a dinucleotide that could then be “flipped” and joined to the other strand, generating a 2 nt palindrome (Lafaille et al., 1989). Alternatively, cleavage at the signal-coding border could be accompanied by formation of a hairpin at the coding end, which could then be nicked close to the terminus, generating a short palindrome (Figure 9). Such a model has been proposed to explain the frequent presence of short inverted duplications at the excision sites of some plant transposons (Coen et al., 1986) and a model for V(D)J recombination incorporating hairpins at coding ends has been proposed (Lieber, 1991). The detection of covalently sealed coding ends in scid thymocytes provides strong physical support for models involving hairpin intermediates. We suggest that in both scid and wild-type cells, hairpins are formed at an early

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The broken molecules identified with a schematic representation locus. Symbols are as in Figure

1

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in this study are diagrammed, along of the relevant region of the TCRG 1 and are not drawn to scale.

Nick hairpins

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step, either during or shortly after the scission of the coding end from the signal heptamer. The hairpin ends are then specifically cleaved by an endonucleolytic activity that introduces a single-strand nick a few nucleotides from the hairpin terminus, generating a short single-strand extension that is complementary to the original end. Retention of one or more nucleotides of the extension after exonucleolytic processing, possibly followed by an N region addition by terminal transferase and subsequent joining, would lead to the characteristic P nucleotide insertion. This explains two key features about P nucleotide inserts isolated from wild-type cells: they are found almost exclusively at intact (nonnibbled) coding ends, and they do not exceed a few nucleotides in length (Lafaille et al., 1989; Aguilar and Belmont, 1991). Although P nucleotides can be found at a substantial fraction of coding joints, they are not universally present. One possibility is that all coding ends go through a hairpin intermediate, but P nucleotides are not always formed. Resolution of hairpins by nicking at varying positions would lead to variation in the number of potential P nucleotides; introduction of a nick between the terminal bases would resolve the hairpin without leading to P nucleotide formation. Potential P nucleotides could also be removed prior to joining, perhaps through the action of exonucleases. One or more of these nucleotides might also be used as short homologies in the joining process, something that would mask their presence (Feeney, 1992). Another possibility is that hairpin termini might be generated at only a fraction of the coding ends. For example, hairpin formation has been observed in the h integrase re action when the normal strand transfer process is blocked; this has been interpreted as a “misguided” attempt by Int to complete strand transfer (Nash and Robertson, 1989). By analogy, it seems possible that the formation of hairpins at coding ends might occur only under certain conditions. Subsequent nicking, either by the recombinase itself or by an accessory factor, could lead to P nucleotide formation. Although further experiments will be required to distinguish among these possibilities, the observation that coding ends are roughly as abundant as signal ends in scid thymocytes suggests that hairpin formation occurs at a substantial fraction of coding termini.

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9. A Hairpin

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of P Nucleotides

According to this scheme, double-strand cleavage occurs at the border between a recombination signal sequence and the adjacent coding region, with hairpin formation at the coding ends. These hairpin structures are resolved by the introduction of specific nicks, in this case 2 nt from the terminus; unwinding generates a single-stranded protrusion whose terminal 2 nt are complementary to the original end. (A specific polarity of nicking is not meant to be implied here; 3’ protrusions are shown for the purpose of illustration.) Optional steps, including exonucleolytic processing and addition of N nucleotides by terminal deoxy nucleotidyl transferase, may occur prior lo joining. Exonucleolytic removal of both complementary nucleotides from the J coding end is illustrated. Joining of the coding ends generates a junction containing two P nucleotides derived from the D coding segment. Symbols are as in Figure 1.

Hairpins and the Role of the scid Factor Experiments using artificial recombination substrates have demonstrated that the frequency of signal joint formation is relatively normal in scid cells, suggesting that the initial cleavage event responsible for liberating signal ends from the coding segments occurs with normal frequency. The results of our physical analysis confirm this prediction, as molecules with signal ends are present in scid thymocytes and the structure of these ends appears grossly similar to signal ends from wild-type cells. In agreement with previous studies (Lieber et al., 1988), production of signal joints appears relatively unaffected, as judged by the presence of circular reciprocal products.

Cell 990

The presence of molecules with hairpin coding ends in scid thymocytes suggests that the scid factor might be involved in the resolution of hairpins. For example, it is possible that an endonucleolytic activity responsible for the introduction of a specific nick adjacent to the hairpin terminus might be absent or impaired in scid cells. In this case, nonspecific mechanisms, such as random nicking or breakage, would be required to “unseal” the hairpin ends. The introduction of occasional, random nicks at variable distances from the termini would account for the rare but very long (up to 15 bp) P nucleotide tracts seen at coding joints from scid mice (Kienker et al., 1991; Schuler et al., 1991). This hypothesis would also explain the high frequency of large deletions observed in coding joints from scid cells, as secondary nicks or breaks might be required to make one or both ends available for joining. If the normal function of the scid factor is to incise DNA near sites of damage (including, but perhaps not limited to, hairpins) the defect in double-strand break repair observed in scid cell lines (Fulop and Phillips, 1990; Biedermann et al., 1991; Hendrickson et al., 1991) might be understandable. An alternative way that thesciddefect might cause accumulation of hairpin coding ends would be through increased production. As stated above, hairpins are formed by the I. integrase reaction when the normal strand transfer process is blocked. It would be conceivable that in scid cells some abnormality of the joining process might cause an unusually high frequency of intramolecular strand transfer, resulting in production of hairpin coding ends at an elevated level. Although this model would be consistent with the high frequency of P nucleotides seen in coding joints from scid thymocytes (Kienker et al., 1991; Schuler et al., 1991), it does not provide a simple explanation for abnormally long P nucleotide inserts nor for the P nucleotides that are frequently found at coding joints in normal cells. A Clue to the Mechanism of Cleavage at Recombination Signals? The identification of hairpins at coding ends may provide information about the mechanism of V(D)J recombination. Other reactions that form DNA hairpins, including sitespecific recombination systems in prokaryotes and yeast (van der Ende et al., 1981; Meyer-Leon et al., 1988; Nash and Robertson, 1989; Reddy and Bauer, 1989; Chen et al., 1992) operate via a mechanism that involves the formation of a covalent DNA-enzyme complex. During cleavage, the protein becomes covalently attached to the DNA, and the energy of the phosphodiester bond is conserved via a phosphoprotein linkage. Misguided attack by this activated complex on the strand opposite the nick leads to hairpin formation (Richet et al., 1988; Nash and Robertson, 1989). Although V(D)J recombination is clearly not a conservative site-specific recombination reaction, the presence of hairpins raises the possibility that covalent DNA-protein complexes might be involved in generating coding ends. Perhaps the initial cleavage event is a nick; an ensuing misguided attack here is the normal pathway, resulting in a double-strand break, with the liberation of a hairpin coding end and a free signal end. According to this

scheme, hairpin formation may be a normal consequence of the mechanism of cleavage, implying that all coding ends may pass through a hairpin stage. The lifetimes of these molecules may be relatively short in normal cells, as they can be incorporated into coding joints; in scid cells, the hairpin species may accumulate as deadend products. Experimental

Procedures

Mice The scid mutation occurred in the C.B-17/ICR (C.B-17) inbred Warn (Bosma et al., 1983); mice homozygous for scid (C.B-17 scidlscid or BALBlc scidlscid [Schuler et al., 1988]) are designated as scid mice; no differences were observed between the two strains. Normal control (wild-type) mice used in this study are BALBlc. Neonatal wild-type mice were used because the abundance of molecules cleaved near TCRS recombination signal sequences IS higher in neonates than in adults (D. B. R., J. P. M.. P. B. N., M. J. B, and M. G.. unpublished data). For scid mice, we used 4-week-old weanlings because thymocyte development is arrested (Carroll and Bosma, 1991) and because the abundance of cleaved molecules does not decrease significantly with age; also, more thymocytes can be obtained from scid weanlings than from neonatal scid mice (D. B. R., J. P. M., P. 8. N., M. J. B., and M. G., unpublished data). scid mice were derived from breeder stocks of the Institute for Cancer Research facility (Fox Chase); wild-type mice were obtained from the National Cancer Institute Division of Cancer Treatment animal facility (National Institutes of Health).

Southern Blot Analysis Genomic DNA preparations were prepared as described (Roth et al., 1992). Restriction enzymes were purchased from Bethesda Research Laboratories and New England BioLabs. Native agarose gel electrophoresis was performed through 25 cm gels; electrophoresis and transfer to Gene Screen Plus nylon membranes (Du Pont) were performed as described previously (Roth et al., 1992). The relative mobilities of size standards (1 kb ladder, Bethesda Research Laboratories) were determined after ethidium bromide starning. For the blot shown rn Figure 6. the samples were electrophoresed through a 25 cm 3% agarose gel consrsting of a 3:l mixture of NuSieve GTG agarose (FMC Corporation) and SeaKem GTG agarose (FMC Corporation) in Trisacetate-EDTA buffer (Sambrook et al., 1989). Alkaline transfer from NuSreve agarose was performed using a POW We pressure blotting apparatus (Stratagene); transfer was carried out in 0.4 N NaOH wrth no pretreatment of the gel. The following protocol was adopted to ensure efficient transfer: 80 min transfer at 50 psi, increased pressure to 60 psi for 60 min, increased pressure to 75 psi for 150 min. Hybridization was carned out in a solutron containing 1% SDS, 1 M NaCI, and 10% dextran sulfate for 14-20 hr, with oligomer-primed (Feinberg and Vogelstern, 1983) “P-labeled probes. The 5 locus hybridization probes used in this work have been described previously (Carroll and Bosma, 1991). DNA fragments used as probes were gel purified prior to labeling.

Two-Dimensional Agarose Gel Electrophoresis Restriction enzyme-digested samples were run in the first dimensron (native) in 0.8% agarose in either Tris-borate-EDTA or Tris-acetateEDTA buffer. The lanes containing the first dimension samples were excused from the gel and equilibrated in alkaline running buffer (50 mM NaOH, 1 mM EDTA). The second dimension (alkaline) was cast so that the gel slice could be sealed with agarose into an extended well. The second dimension was run in 1% agarose (11 cm x 14 cm), generally at 30 V for 8 hr, with a glass plate over the gel and buffer recirculation. The DNA was then transferred to Gene Screen Plus under alkaline conditions (0.4 N NaOH) for 1 hr using a positive pressure blothng apparatus. Hybridization was carried out as described above. In the Sl nuclease experiment (Figure 4C), approximately 7 ug of EcoRIdigested adult scid thymus DNA was treated with 5 U of Sl nuclease (Bethesda Research Laboratories) for 10 min under conditions recommended by the manufacturer.

Hairpin 991

Coding

Ends

in scid Thymocytes

Exonuclease Assays Undigested genomic DNA (40-100 frg per assay) was treated with 0.75-l .5 U of M. luteus ATP-dependent DNAase (US Biochemical) for 4 hr at 37% in a reaction buffer containing 66 mM glycine (pH 9.4). 30 mM MgCI,, 8.3 mM j%mercaptoethanol, and 0.5 mM ATP. After incubation, the DNA was purified by phenol extraction and ethanol precipitation, resuspended, and digested with the appropriate restriction enzyme prior to electrophoresis. Acknowledgments We thank Regis Krah, Fraser McBlane. Howard Nash, Sharon Roth, Moshe Sadofsky, and Cece Trainor for critically reading the manuscript. The insights, critical suggestions, and enthusiasm contributed by Tania Baker and Kiyoshi Mizuuchi are gratefully acknowledged. This work was supported in part by National Institutes of Health (NIH) grants Al-13323, CA-04946, and RR-05539 and by an appropriation from the Commonwealth of Pennsylvania. J. P. M. was supported by a National Research Council-NIH research associateship. D. B. R. is a Howard Hughes Medical Institute Physician Research Fellow. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenl’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received

June

8, 1992; revised

Kienker, L. J., Kuziel, W. A., Garni-Wagner, B. A., Kumar, V., and Tucker, P. W. (1991). T cell receptor y and S gene rearrangements in scidthymocytes: similarity to those in normal thymocytes. J. Immunol. 147, 4351-4359. Lafaille, J. J., DeCloux, A., Bonneville, M., Takagaki, Y., and Tonegawa, S. (1989). Junctional sequences of T cell receptor 76 genes: implications for y6 T cell lineages and for a novel intermediate of V-(D)-J joining. Cell 59, 859-870. Lewis, S., and Gellert, M. (1989). The mechanism gene assembly. Cell 59, 585-588.

of antigen

Lieber, M. R. (1991). Site-specific tem. FASEB J. 5,2934-2944.

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recombination

receptor sys-

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The inverted repeat as a recognizable structural DNA molecules. Proc. Natl. Acad. Sci. USA 77,

McCormack, W.T.,Tjoelker, L. W.,Carlson, L. M., Petryniak, B., Barth, C. F., Humphries, E. H., andThompson, C. B. (1989). Chicken lgLgene rearrangement involves deletion of a circular episome and addition of single nonrandom nucleotides to both coding segments. Cell 56, 785791.

July 21, 1992

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