Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation - PubMed (original) (raw)

Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation

M A Bogue et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Previous studies have suggested that ionizing radiation causes irreparable DNA double-strand breaks in mice and cell lines harboring mutations in any of the three subunits of DNA-dependent protein kinase (DNA-PK) (the catalytic subunit, DNA-PKcs, or one of the DNA-binding subunits, Ku70 or Ku86). In actuality, these mutants vary in their ability to resolve double-strand breaks generated during variable (diversity) joining [V(D)J] recombination. Mutant cell lines and mice with targeted deletions in Ku70 or Ku86 are severely compromised in their ability to form coding and signal joints, the products of V(D)J recombination. It is noteworthy, however, that severe combined immunodeficient (SCID) mice, which bear a nonnull mutation in DNA-PKcs, are substantially less impaired in forming signal joints than coding joints. The current view holds that the defective protein encoded by the murine SCID allele retains enough residual function to support signal joint formation. An alternative hypothesis proposes that DNA-PKcs and Ku perform different roles in V(D)J recombination, with DNA-PKcs required only for coding joint formation. To resolve this issue, we examined V(D)J recombination in DNA-PKcs-deficient (SLIP) mice. We found that the effects of this mutation on coding and signal joint formation are identical to the effects of the SCID mutation. Signal joints are formed at levels 10-fold lower than in wild type, and one-half of these joints are aberrant. These data are incompatible with the notion that signal joint formation in SCID mice results from residual DNA-PKcs function, and suggest a third possibility: that DNA-PKcs normally plays an important but nonessential role in signal joint formation.

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Figures

Figure 1

Figure 1

Thymocytes from SLIP mice are defective in TCR Vβ8-Jβ2 coding joint formation. Thymocyte DNA samples were subjected to semiquantitative PCR analysis. Titrations with wild-type DNA (100, 10, 1, 0.1 ng; lanes 1–4, respectively) were performed to estimate the abundance of coding joint formation in SCID (lane 5) and two SLIP (lanes 6 and 7) DNA samples (all at 100 ng). Our primers amplify Vβ8 rearrangements to all Jβ2 members. The most prominent band in the wild-type DNA lanes is 276 bp, the expected size for Vβ8-Jβ2.6 (unrearranged DNA is not amplified). A negative PCR control (all reagents without DNA) is shown in lane 8. Relevant sizes of the DNA marker (1-kb ladder; GIBCO/BRL) are indicated adjacent to lane M.

Figure 2

Figure 2

IgH recombination products in SLIP mice. (A) Coding joints (D–J) and hybrid joints (5′ D recombination signal sequence to J) are amplified from the same primers (horizontal arrows). Coding elements are depicted by boxes and recombination signal sequences are represented by triangles adjacent to the coding elements. Both types of joints depend on RAG-mediated cleavage at the recombination signal sequence 5′ of the J coding element. Normal coding joints are generated when cleavage also occurs 3′ of the D coding element (open vertical arrows) and hybrid joint formation depends on cleavage on the 5′ side of this element (solid vertical arrows). (B) Coding joint formation is defective in slip bone marrow. Bone marrow DNA samples were subjected to semiquantitative PCR analysis; titrations with wild-type DNA (100, 10, 1, 0.1 ng; lanes 1–4, respectively), SCID (lane 5), and two SLIP (lanes 7 and 9) DNA samples (all at 100 ng). Both coding (cj) and hybrid joints (hj) are detected by a JH4-specific probe (Upper); expected sizes are ≈100 bp for coding joints [numerous sizes caused by amplification of several D region family members by a degenerate primer (DHL)] and 83 bp for a perfect hybrid joint. The membrane was stripped and hybridized to an oligonucleotide probe representing a perfect hybrid joint (Lower). Relevant sizes of the DNA marker (lane M) are indicated.

Figure 3

Figure 3

Privileged site coding joint formation in SCID mice does not depend on residual DNA-PKcs functions. Thymocyte DNA samples were subjected to semiquantitative PCR analysis for TCR Dδ2-Jδ1 coding joints. Titrations with wild-type DNA (100, 10, 1, 0.1 ng; lanes 1–4, respectively) were performed to estimate the abundance of coding joint formation in SCID (lane 5) and three SLIP (lanes 6–8) DNA samples (all at 100 ng). The expected size is 199 bp. A negative PCR control (all reagents without DNA) is shown in lane 9. Relevant sizes of the DNA marker are indicated adjacent to lane M.

Figure 4

Figure 4

Signal joint formation in SLIP mice. (A) TCR Dδ2-Jδ1 signal joints were amplified from thymocyte DNA preparations (the same DNA concentrations as in Fig. 3): wild-type titration (lanes 1–4), SCID (lane 6), and SLIP (lane 7). The expected size is 301 bp. A negative PCR control (all reagents without DNA) is shown in lane 8. Relevant sizes of the DNA marker (lane M) are indicated. (B) Signal joint PCR products were subjected to digestion with _Apa_LI. Wild type (lanes 1 and 2), SCID (lanes 3 and 4), and SLIP (lanes 5 and 6); undigested (lanes 1, 3, and 5), and _Apa_LI treated (lanes 2, 4, and 6). The expected sizes after digestion are 167 and 134 bp; only the larger product hybridizes to the probe.

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