Activation of V(D)J Recombination Induces the Formation of Interlocus Joints and Hybrid Joints in scid Pre-B-Cell Lines

Abstract

V(D)J recombination is the mechanism by which antigen receptor genes are assembled. The site-specific cleavage mediated by RAG1 and RAG2 proteins generates two types of double-strand DNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although these DNA breaks are mainly resolved into coding joints and signal joints, they can participate in a nonstandard joining process, forming hybrid and open/shut joints that link coding ends to signal ends. In addition, the broken DNA molecules excised from different receptor gene loci could potentially be joined to generate interlocus joints. The interlocus recombination process may contribute to the translocation between antigen receptor genes and oncogenes, leading to malignant transformation of lymphocytes. To investigate the underlying mechanisms of these nonstandard recombination events, we took advantage of recombination-inducible cell lines derived from scid homozygous (s/s) and scid heterozygous (s/؉) mice by transforming B-cell precursors with a temperature-sensitive Abelson murine leukemia virus mutant (ts-Ab-MLV). We can manipulate the level of recombination cleavage and end resolution by altering the cell culture temperature. By analyzing various recombination products in scid and s/؉ ts-Ab-MLV transformants, we report in this study that scid cells make higher levels of interlocus and hybrid joints than their normal counterparts. These joints arise concurrently with the formation of intralocus joints, as well as with the appearance of opened coding ends. The junctions of these joining products exhibit excessive nucleotide deletions, a characteristic of scid coding joints. These data suggest that an inability of scid cells to promptly resolve their recombination ends exposes the ends to a random joining process, which can conceivably lead to chromosomal translocations.Developing lymphocytes have the unique ability to generate diverse antigen receptor molecules, immunoglobulins, and T-cell receptors. The assembly of these receptor genes is achieved through site-specific recombination events from separately encoded gene segments, variable (V), diversity (D), and joining (J) regions, in a process known as V(D)J recombination (6). Each gene segment is flanked by conserved recombination signal sequences (RSS) with a spacer of 12 or 23 nucleotides (12-RSS or 23-RSS, respectively). V(D)J recombination, catalyzed by enzymes encoded by recombination-activating genes 1 and 2 (RAG1 and RAG2), takes place at the junctions between RSS and coding gene segments (5, 19). Cleavage occurs coordinately at 12-RSS and 23-RSS, in accordance with what is known as the 12/23 rule. This site-specific cleavage generates two types of broken DNA molecules: bluntopened signal ends and covalently sealed coding ends (22, 39).The joining of these ends to form signal joints (SJ) and coding joints (CJ) is mediated by nonhomologous end joining machinery, which is believed to be the principal pathway to repair double-stranded breaks (DSBs) in vertebrate cells (29,30). Identificat...

V(D)J recombination in zebrafish: Normal joining products with accumulation of unresolved coding ends and deleted signal ends

“…As shown in Figure 2B, both SEs and SJs made in Sp9 are sensitive to ApaL1 digestion, indicating the presence of intact SEs and précised joining of these ends in wild-type cells. On the other hand, S9 cells produce intact SEs but imperfect SJs (because of incomplete ApaL1 digestion), which results from their partial abnormality in SJ formation, as reported previously (Lew et al, 2000). In contrast, the integrity of SEs and SJs recovered from zebrafish samples was found inverse to those made in S9 cells, i.e.…”

Identification of an aberrant type of rearrangement in the T-cell receptor α/δ locus in adult T-cell leukemia

“…Lew et al (2000) have demonstrated that severe combined immude deficiency (scid)-homozygous (s/s-ts) pre-B-cell lines transformed with a temperature-sensitive Abelson murine leukemia virus mutant (ts-Ab-MLV) produced high levels of hybrid joints, exceeding that of its scid-heterozygous (s/ϩ-ts) counterparts expressing functional DNA-PKcs. The s/ϩ-ts cells mostly produced hybrid joints with an intact RSS and coding sequence, while the s/s-ts cells exhibited hybrid joints with extensive nucleotide deletions in both the RSS and the coding sequence.…”

V(D)J recombination of chromosomally integrated, wild-type deletional and inversional substrates occur at similar frequencies with no preference for orientation

“…The data suggest that RAGs, HMGB1, DNAPKcs, Ku70, and Ku80 form a pre-cleavage complex on the RSS. This explains the high incidence of non-standard [44,45,52,53,80] and interlocus joints [54,81] formed in scid cells.…”

“…The scid mutation is mapped to the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) [49][50][51]. Scid cells exhibit a high frequency of non-standard junction formation [52][53][54] and thus, are useful for detection of nonstandard junctions in our assays. Our results indicate that murine pre-B cell do not prefer the wild-type 12 bp RSS in the deletional orientation over the inversional orientation.…”