What prevents intermolecular V(D)J recombination?

Abstract

Generation of a highly diverse repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) molecules is ofparamount importance to the immune system. This task isaccomplished through a site-specific DNA rearrangement mechanism, V(D)J recombination, that assemblesthe genes encoding the antigen-binding regions of theseimmune receptor molecules from a large library of genesegments (termed V, D, and J elements) that are separatedin the germ line by variable lengths of noncoding DNA.The same mechanism is used for rearrangement of Iggenes (in developing B lymphocytes) and TCR genes (indeveloping T lymphocytes). Each gene segment isflanked by a recombination signal sequence (RSS), whichconsists of conserved heptamer and nonamer elementsseparated by either 12 or 23 nucleotides of nonconservedDNA sequence. Efficient rearrangement requires oneRSS of each spacer length (a restriction termed the 12/23rule) (Eastman et al. 1996; Steen et al. 1996; van Gent etal. 1996). Rearrangement is initiated by binding of the recombinase proteins, RAG-1 and RAG-2, to the RSS (Fig.1). Formation of a DNA-protein complex containing theRAG proteins bound to a 12-23 RSS pair (a synapticcomplex) is followed by DNA cleavage, which introduces double-strand breaks (DSBs) precisely betweeneach RSS and its adjacent coding element. Cleavage produces two types of broken ends: blunt signal ends, whichterminate in the RSS, and covalently closed (hairpin)coding ends. Joining of these ends requires participationof the machinery used for DSB repair, including components of the DNA-dependent protein kinase (DNA-PK),and generates two types of recombinant junctions: signaljoints and coding joints, respectively (for review, seeLewis 1994; Grawunder et al. 1998). In vivo experimentssuggest that after cleavage, all four ends remain associated in a postcleavage complex (Lewis et al. 1988; Zhu etal. 1996). However, recent in vitro work suggests that thesignal ends are more stably bound to the RAG proteinsthan the coding ends (Hiom and Gellert 1998). The stepsinvolved in end processing and joining and the roles ofDSB repair factors remain unclear. In vivo studies suggest that opening of the hairpin coding ends requires several DSB repair factors, including Ku86 and the catalyticsubunit of the DNA-PK, because hairpin-coding ends accumulate in mutant lymphocytes lacking these proteins(Roth et al. 1992; Zhu and Roth 1995; Zhu et al. 1996).The RAG proteins themselves may also play a part in thejoining of coding ends. Both in vitro analysis (Leu et al.1997; Ramsden et al. 1997) and analysis of mutant RAGproteins in vivo (Steen et al. 1999) indicate that the RAGproteins stimulate formation of coding joints. Furthermore, recent work suggests that the RAG proteins are capable of opening hairpins (Besmer et al. 1998). Thus,both the RAG proteins and components of the DSB repairmachinery may participate in coding joint formation...