DNA Hairpin Opening Mediated by the RAG1 and RAG2 Proteins

Abstract

The lymphoid cell-specific proteins RAG1 and RAG2 initiate V(D)J recombination by cleaving DNA adjacent to recombination signals, generating blunt signal ends and covalently sealed, hairpin coding ends. A critical next step in the reaction is opening of the hairpins, but the factor(s) responsible has not been identified and had been thought to be a ubiquitous component(s) of the DNA repair machinery. Here we demonstrate that RAG1 and RAG2 possess an intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip. In Mn 2؉, a synthetic hairpin is nicked 5 nucleotides (nt) 5 of the hairpin tip, with more distant sites of nicking suppressed by HMG2. In Mg 2؉ , hairpins generated by V(D)J cleavage are nicked whereas synthetic hairpins are not. Cleavage-generated hairpins are nicked at the tip and predominantly 1 to 2 nt 5 of the tip. RAG1 and RAG2 may therefore be responsible for initiating the processing of coding ends and for the generation of P nucleotides during V(D)J recombination.V(D)J recombination assembles the variable regions of antigen receptor genes during lymphocyte development by joining together V (variable), J (joining), and in some cases D (diversity), coding gene segments (28). Recombination is specifically directed to coding elements by recombination signal sequences (RSSs) which flank the segments to be joined. These RSSs consist of a conserved heptamer which is contiguous to the coding flank and an AT-rich nonamer. Heptamer and nonamer are separated by a nonconserved spacer of either 12 or 23 bp, yielding the 12-RSS or 23-RSS, respectively. In vivo, recombination primarily occurs between coding elements with RSS spacers of different lengths, thereby preventing the joining of inappropriate elements. This restriction is referred to as the 12/23 rule.Mechanistically, the recombination reaction is envisioned to occur in two stages. In the first stage, initiation of recombination is mediated by the proteins encoded by the recombinationactivating genes, RAG1 and RAG2, which bind directly to RSSs (10,32,47,51). Binding by RAG proteins is followed by RSS synapsis and concerted cleavage at both signals. Cleavage involves hydrolytic nicking at the heptamer-coding flank border, and the the 3Ј-hydroxyl thus generated serves as a nucleophile to attack the phosphodiester bond on the other DNA strand opposite the nick in a direct transesterification reaction (32, 52). The coding ends generated by cleavage are covalently sealed DNA hairpins, while signal ends are blunt and 5Ј phosphorylated. This reaction is stimulated in vitro, especially at the 23-RSS, by addition of DNA-bending proteins HMG1 and HMG2 (44,50). Coordinate cleavage in accordance with the 12/23 rule requires Mg 2ϩ rather than Mn 2ϩ as divalent metal cofactor and is stimulated by HMG1 or HMG2 (12,43,44,50,53). In the second stage of the reaction, coding ends are processed, and coding joints and signal joints form in reactions with similarities to the repair of DNA double-strand breaks by nonhomologo...

DNA structures at chromosomal translocation sites

“…(71) The RAG cleavage on non-B DNA structures occurs under physiologic (in the presence of Mg 2þ ) buffer conditions. (62) The earlier reports that RAGs are capable of cleaving 3 0 overhangs (72,73) and of cleaving 3 0 flaps (73) have more significance in light of our recent findings (Fig.…”

Metabolism of Recombination Coding Ends in scid Cells

“…The purified RAG1/2 proteins were found to mediate the reverse reaction of the cleavage to generate hybrid joints and open-shut products (31). Recently, two laboratories have shown that RAG1/2 proteins can nick hairpin ends (29,30). Interestingly, however, the resulting opened ends contained 5Ј overhangs, which differ from the opened coding ends made in vivo (Table I; 9).…”

“…It has been shown that RAG1/2 proteins can nick artificial hairpin ends in a cell-free system (29,30). To determine whether these proteins are responsible for processing hairpin ends in vivo, we analyzed RAG2 expression in various cell samples.…”

“…RAG1 and RAG2 proteins have been shown to possess end-processing activities, including binding of recombination intermediates (27,28), nicking synthetic hairpin ends (29,30), and rejoining cleaved signal ends to coding ends (31). Recently, it has been demonstrated that the Mre11 protein, when complexed with Rad50 and the Nijmegen breakage syndrome gene product, Nbs1, exhibits several nuclease activities in vitro, such as hairpin nicking and processing of opened ends (32).…”

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

“…DNA hairpin opening activities have been observed in vitro for the Mre11/Rad50/Nbs1 complex (29), which functions in the cellular response to DNA damage (30,31) for the RAG1/RAG2 proteins (32,33) and most recently, for the Artemis protein (34). Because the SCID DNA-PK deficiency is not known to affect the expression of any of these proteins, it was of interest to re-examine whether a portion of coding ends are open in SCID mice.…”