Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase

Abstract

The RAG1 and RAG2 proteins collaborate to initiate V(D)J recombination by binding recombination signal sequences (RSSs) and making a double-strand break between the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins, the bacterial transposases and retroviral integrases, cleavage by the RAG proteins requires a divalent metal ion but does not involve a covalent protein/DNA intermediate. In the transposase/integrase family, a triplet of acidic residues, commonly called a DDE motif, is often found to coordinate the metal ion used for catalysis. We show here that mutations in each of three acidic residues in RAG1 result in mutant derivatives that can bind the RSS but whose ability to catalyze either of the two A series of site-specific somatic DNA rearrangements is required for the assembly of the immunoglobulin and T-cell receptor (TCR) genes of the vertebrate immune system (for reviews, see Lewis 1994;Gellert 1997;Oettinger 1999). These V(D)J recombination events are initiated by the combined action of two lymphoid-specific proteins, RAG1 and RAG2. Together, these proteins recognize recombination signal sequences (RSSs) that flank immunoglobulin and TCR gene segments and introduce a double-strand break (DSB) at the border of the coding gene segment and the RSS. The RSS is comprised of conserved heptamer and nonamer elements separated by a spacer region of conserved length (12 or 23 bp) but variable sequence. The DSB is generated in two steps (McBlane et al. 1995). In the first step, a nick is introduced at the 5Ј end of the heptamer at the signal/coding boundary. In the second step, the newly liberated hydroxyl on the coding DNA attacks the phosphodiester bond of the opposing strand to form a hairpinned coding end and a blunt signal end. Subsequent processing and rejoining of the cleaved ends to form the mature coding segment and a signal junction requires the action of a number of proteins, including several involved in the repair of X-rayinduced damage (Jeggo 1998).The reactions carried out by the RAG proteins are chemically similar to those carried out by the transposase proteins of bacterial transposons such as Mu or Tn10 or by the integrase proteins of retroviruses such as HIV (Plasterk 1998;Roth and Craig 1998). In addition to carrying out the two steps of V(D)J cleavage, the RAG proteins can mediate transpositional recombination in vitro, joining the 3Ј OH of a cleaved signal end to an unrelated target sequence (Agrawal et al. 1998;. A shared feature of the strand transfer reactions of these mobile elements and the RAG recombinase is that they occur in the absence of an external energy source via a one-step transesterification reaction with no protein-DNA intermediate (van Gent et al. 1996). One distinction between the RAG proteins and other recombinases is the reversal of the initial cleavage site relative to the DNA recognition sequence. Specifically, RAG proteins nick the top strand (the 5Ј end of the heptamer at the signal/coding boundary), whereas MuA and Tn10 transposases, for...

Identification and Characterization of a Gain-of-Function RAG-1 Mutant

“…In support of this possibility, we report here that mutation of a single residue in RAG-1, E649A, specifically enhances the ability of the RAG complex to catalyze hairpin formation in vitro and facilitate greater cleavage of plasmid V(D)J recombination substrates containing an unpaired RSS or a mispaired (12/12 or 23/23) RSS in vivo, in violation of the 12/23 rule. The E649A RAG-1 mutation is located within the central domain of RAG-1 (8), which encompasses two residues critical for the catalytic activity of the recombinase, D600 and D708 (12,21,24). This colocalization suggests that the central domain plays an active role not only in recognition and catalysis at the site of DNA cleavage but also in perceiving 12/23-regulated synapsis.…”

“…These studies have defined regions of RAG-1 and RAG-2 dispensable for supporting the rearrangement of plasmid V(D)J recombination substrates in cell culture (7,22,43,44); identified various mutations in the RAG proteins that impair RSS binding (9,11,49,52), one or both cleavage steps (12,20,24,28,39), or the joining phase of V(D)J recombination (21,39,47,54); and refined structural elements required for RAG-1/RAG-2 association or interactions with other proteins (2,33,60). RAG mutants that enhance the cleavage or joining phases of V(D)J recombination have not yet been reported, although mutations in RAG-2 that impair its degradation at the G 1 /S transition of the cell cycle enable the RAG complex to cleave DNA beyond the G 0 to G 1 phases, where it is normally restricted (25).…”

The PHD domain of the sea urchin RAG2 homolog, SpRAG2L, recognizes dimethylated lysine 4 in histone H3 tails

“…While the active site of the V(D)J recombinase resides entirely in RAG1, [7][8][9], RAG2 acts as an essential accessory factor enhancing the recognition of the RSS and the stability of the RAG1/2-RSS complex [10][11][12][13]. RAG2 can conceptually be divided into two regions, the N-terminal core region (murine amino acids 1-383) essential for all catalytic activities, and the C-terminal non-core region (murine amino acids 384-527) dispensable for recombination on artificial DNA substrates [ 14,15 ].…”

Requirements for DNA hairpin formation by RAG1/2

“…2 for a review). Although no structural information exists for the core RAG regions, biochemical data has identified the binding site in RAG1 for the catalytic metal ions coordinated by three acidic residues (D600, D708, and E962) (14)(15)(16). These sites are likely to be held in an RNaseH-like fold also possessed by transposases (17).…”