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. 1998 Apr;18(4):2029-37.
doi: 10.1128/MCB.18.4.2029.

Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination

M S Schlissel  1
Affiliations

Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination

M S Schlissel. Mol Cell Biol. 1998 Apr.

Abstract

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.

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Figures

FIG. 1
FIG. 1
V(D)J recombination reaction pathway and LM-PCR assay for reaction intermediates. (A) Diagram of the reactants (top), broken DNA intermediates (middle), and products (bottom) of V(D)J recombination. V and J gene segments, with their associated RSSs (heptamer [H] and nonamer [N]) are recognized and cleaved by the recombinase at the RSS–coding-segment junction (arrow), generating coding-end and signal-end fragments. These ends are joined to form a coding joint and a signal joint. (B) LM-PCR assay for broken-ended recombination reaction intermediates. The BW linker is ligated to available ends in total genomic DNA by using T4 DNA ligase. The sites of linker ligation are revealed by a set of nested PCR assays with a linker primer (BW-1) and locus-specific primers (open arrows labeled 1, 2, 4, and 5). Blots of PCR products were probed with internal oligonucleotides (solid lines labeled 3 and 6).
FIG. 2
FIG. 2
Broken coding-end DNA can be detected in induced 103 bcl2/4 cell DNA. (A and B) 103 bcl2/4 cells were induced to activate V(D)J rearrangement by a temperature shift. DNA was prepared from induced 39°C (lanes +) and uninduced 33°C (lanes −) samples by the indicated techniques as described in the text. The samples were analyzed by LM-PCR for broken signal ends (se) (A) and broken coding ends (ce) (B) at the Jκ1 locus. Phosphorimages of oligonucleotide-probed Southern blots of reaction products are shown with the positions of various signal and coding ends indicated by arrows. (C) Ethidium bromide-stained agarose gel analysis of control amplifications of a nonrearranging locus (CD14) from the same DNA samples used in panels A and B. Controls included buffer in place of template (BL; lane 8) and 63-12 cell DNA (a RAG-2-deficient cell line; lane 9). Lanes 6 and 7 contain two independently purified DNA samples. SDS-Pk, sodium dodecyl sulfate plus proteinase K.
FIG. 3
FIG. 3
T4 DNA polymerase treatment enhances LM-PCR detection of broken coding ends. (A to C) DNA samples prepared by the agarose plug method from uninduced (33°C [33 degr]) and induced (39°C [39 degr]) 103 bcl2/4 cells (A and B) and from newborn thymus (C) were analyzed by LM-PCR for broken Jκ1 coding (A), Jκ1 and Jκ2 signal (B), and JH2 coding and signal (C) ends without (lanes −) or with (lanes +) T4 DNA polymerase (T4 pol) pretreatment. Controls included identically prepared and treated 63-12 (RAG-2-deficient) cell DNA and buffer (lane C). DNA samples from panel C were amplified with primers specific for a nonrearranging genomic locus, demonstrating the presence of DNA in all samples. (D) Ethidium bromide-stained agarose gel of these control amplifications. Lanes 1 to 4 correspond to samples 1 to 4 in panel C, lanes 5 to 8 correspond to samples 6 to 9 in panel C, and lane 9 is a buffer-only control amplification. Lanes 1 to 6 of panels A and B were shown to contain equivalent amounts of DNA by a similar method (data not shown). ce, coding ends; se, signal ends.
FIG. 4
FIG. 4
DNA sequence analysis of cloned broken coding- and signal-end LM-PCR fragments. Broken coding-end fragments were excised from agarose gels, reamplified, purified, and cloned into the vector pBSK. Randomly selected coding-end clones were sequenced. The arrows above the germ line sequences indicate sites of linker ligation determined by sequencing. The numbers above the arrows indicate the frequencies of individual sequences from sets of consecutive sequences. The solid vertical bar indicates the RSS–coding-segment junction, with arrows to the right indicating coding ends and those to the left indicating signal ends. The RSS heptamer and nonamer sequences are boxed. Signal-end sequences were reported previously and are shown for comparison only (28).
FIG. 5
FIG. 5
Length heterogeneity of amplified coding ends and joints. DH, JH1, JH2, Vκ, and Jκ1 coding ends (ce) (A, B, C, E, and F) and DJH and VJκ joints (D and G) were amplified from T4 polymerase-treated thymus or induced 103 bcl2/4 cell DNA. The amplified fragments were gel purified and end labeled with [γ-32P]ATP by using T4 DNA kinase. Labeled fragments were electrophoresed on 6% denaturing polyacrylamide gels alongside DNA sequencing ladders used as size markers. The diagrams adjacent to each coding-fragment gel image indicate the sequence position based on these comigrating sequence markers. Position zero in the diagrams corresponds to the full-length coding end (i.e., the junction between the RSS and the coding segment), positive numbers indicate coding-end deletions, and negative numbers indicate longer-than-full-length coding-end lengths. The diagrams adjoining DJH and VJκ joints indicate successive nucleotide lengths. In panel E, the lane labeled Vκ mkr contains a radiolabeled amplification product of genomic DNA demonstrating the length heterogeneity of intact Vκ genes (see the text).
FIG. 6
FIG. 6
Jκ1 and JH2 coding ends have 3′ overhangs. (A and B) DNA purified in agarose plugs from newborn-mouse thymocytes (A) or 103 bcl2/4 cells cultured at 33 or 39°C (B) (shift − or +) was ligated to the BW linker without pretreatment (lanes none) or after treatment with either T4 DNA polymerase (lanes T4 Pol) or mung bean nuclease (lanes MB-N). Ligated plugs were analyzed by PCR for JH2 (A) or Jκ1 (B) coding-end (ce) breaks (arrows). The lanes labeled 63-12 were control LM-PCR assays with 63-12 cell DNA. Lanes 1 and 2 in panel A represent independent thymocyte DNA samples. (C) Amplified products from lanes 1, 3, and 4 in panel A and lanes 2, 4, and 6 in panel B were gel purified, reamplified for five cycles with a 32P-labeled specific oligonucleotide, and analyzed by denaturing polyacrylamide gel electrophoresis. In each case, the arrow indicates the predominant broken-ended molecule (+9 for JH2 and +4 for Jκ1) and the tick marks indicate 1-nt intervals (determined by the electrophoresis of a DNA sequencing reaction mixture on the same gel). Lanes: N, no pretreatment; T, T4 DNA polymerase pretreatment; M, mung bean nuclease pretreatment.
FIG. 7
FIG. 7
Analysis of nonblunt coding ends by LM-PCR with 5′- and 3′-overhanging linkers. (A) Structure of linkers designed to ligate to 3′- and 5′-overhanging DNA breaks. The asterisks indicate the nucleotides directly ligated in each case. N indicates an equimolar mixture of each deoxynucleoside triphosphate. cbe, coding broken end. (B) LM-PCR assays testing the ability of blunt (lanes A), 3′-overhanging (lanes B and C), or 5′-overhanging (lanes D and E) linkers to detect blunt-ended (lanes 1 to 5), uncut (lanes 6 to 11), 5′-overhanging (lanes 12 to 17), or 3′-overhanging (lanes 18 to 22) plasmid targets mixed with genomic DNA. An ethidium-stained agarose gel of PCR products is shown. Linkers B and D have 2-nt degenerate overhangs, and linkers C and E have 4-nt degenerate overhangs. (C) Jκ1 coding breaks detected with blunt (lanes B) or 3′- or 5′-overhanging linkers (lanes 3′ and 5′). The overhanging linkers were equimolar mixtures of linkers with 2-, 3-, and 4-nt overhangs. CD19+ bone marrow B-cell DNA, uninduced or induced 103 bcl2/4 cell DNA, and 63-12 cell DNA in agarose plugs were ligated with the indicated linkers and subjected to PCR with primers specific for the Jκ1 coding-end break. A phosphorimage of an oligonucleotide-probed Southern blot is shown, with the arrow indicating the position of migration of the Jκ1 coding-end break (ce). (D) JH2 coding-end breaks detected with linkers as indicated for panel C. DNA in agarose plugs purified from two separate thymocyte preparations (lanes 1 to 3 and lanes 4 to 6) or 63-12 cells (lanes 7 to 9) was ligated to the indicated linkers and subjected to PCR with primers specific for the JH2 coding-end break. A phosphorimage of an oligonucleotide-probed Southern blot is shown, with the arrow indicating the position of migration of the JH2 coding-end break (ce). (E) Control PCR assays on DNA samples used in each assay in panels C and D. An ethidium-stained gel analysis is shown, with the arrow indicating the position of the specific CD14 gene amplification product. (F) Denaturing polyacrylamide gel analysis of LM-PCR products generated from T4 DNA polymerase-polished linker-ligated (lanes T4) or 3′-overhanging linker-ligated (lanes 3′) amplified 103 bcl2/4 cell or thymus DNA, demonstrating the range of sizes of the PCR products.
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