Metabolism of Recombination Coding Ends in scid Cells

Abstract

V(D)J recombination cleavage generates two types of dsDNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although signal ends can be directly ligated to form signal joints, hairpin coding ends need to be opened and subsequently processed before being joined. However, the underlying mechanism of coding end resolution remains undefined. The current study attempts to delineate this process by analyzing various structures of coding ends made in situ from recombination-inducible pre-B cell lines of both normal and scid mice. These cell lines were derived by transformation of B cell precursors with the temperature-sensitive Abelson murine leukemia virus. Our kinetic analysis revealed that under conditions permissive to scid transformants, hairpin coding ends could be nicked to generate 3′ overhangs and then processed into blunt ends. The final joining of these blunt ends followed the same kinetics as signal joint formation. The course of this process is in sharp contrast to coding end resolution in scid heterozygous transformants that express the catalytic subunit of DNA-dependent protein kinase, in which hairpin end opening, processing, and joining proceeded very rapidly and appeared to be closely linked. Furthermore, we demonstrated that the opening of hairpin ends in scid cells could be manipulated by different culture conditions, which ultimately influenced not only the level and integrity of the newly formed coding joints, but also the extent of microhomology at the coding junctions. These results are discussed in the context of scid leaky recombination.

Variable Diversity Joining Recombination: Nonhairpin Coding Ends in Thymocytes of SCID and Wild-Type Mice

“…Although temperature-dependent opening of hairpin coding ends was recently reported in a bcl-2 transgenic SCID pre-B cell line transformed by a temperature-sensitive Abelson murine leukemia virus (ts-Abl-MLV) (46), the present work is first to demonstrate open coding ends in unmanipulated SCID lymphocytes. In this study, the portion of SCID 5ЈD␦2 coding ends that were open was estimated to be ϳ25%.…”

“…Open coding ends obtained from DNA-PKcs null mice appeared qualitatively and quantitatively similar to those from SCID, negating the possibility that partial function of the truncated DNA-PKcs in SCID allows some hairpin nicking. It should be emphasized that 5Ј overhanging coding ends were not identified after temperature-dependent opening of hairpin coding ends in ts-Abl-MLV-transformed SCID pre-B cells (46). Depending on cell culture conditions, these transformants predominantly generated either blunt coding ends with minor deletions or staggered ends with 3Ј overhangs that were progressively deleted with extended culture at the nonpermissive temperature.…”

Role of poly(ADP-ribosyl)ation in DNA-PKcs- independent V(D)J recombination

“…The lack of pADPr foci in s͞ϩ-ts cells is consistent with rapid resolution of coding ends and͞or containment of the recombination intermediates within the RAG complexes. However, in the scid-ts cells, coding end processing occurs upon RAG down-regulation and thus it is rather unlikely that coding end processing occurs within the RAG complex (24). Therefore, it is probable that once the RAG proteins dissociate from the recombination ends, PARP-1 recognizes and binds to protect the exposed ends, in essence forming a new DNA-protein complex.…”

“…In an effort to overcome this limitation and to decipher the end processing events at endogenous antigen receptor gene loci, we developed a scid-ts pre-B cell line that provides the opportunity to analyze each reaction in a piecemeal fashion (21). As described in detail (21,24), at the permissive temperature (33°C), RAG expression is low, Ig light chain (IgL-chain) loci are inaccessible, and thus a very low level of recombination occurs. At the nonpermissive temperature (39°C), RAG expression is up-regulated and the IgL-chain loci become accessible, resulting in recombination initiation.…”

“…However, the hairpin coding ends of the scid-ts cells appear to be inaccessible to the nicking enzyme as they persist at the nonpermissive temperature. When returned to the permissive temperature (39°C333°C, postinduction), the hairpin ends are made accessible to a nicking enzyme resulting in the appearance of stable, opened coding ends, which is followed by their religation to form coding joints (24).…”