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

Abstract

RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis. V(D)J recombination is the process by which noncontiguous antigen receptor gene coding segments, called variable (V), diversity (D), and joining (J), are assembled during lymphocyte development to produce the variable region exon of a mature antigen receptor gene (3). V(D)J recombination occurs in two distinct phases. In the first phase, two lymphoid cell-specific proteins called RAG-1 and RAG-2 assemble a multiprotein synaptic complex with two different gene segments through interactions with a conserved recombination signal sequence (RSS) that adjoins each gene segment. Each RSS contains a conserved heptamer and nonamer sequence, separated by either 12 or 23 base pairs of intervening DNA of more varied composition (12-RSS and 23-RSS, respectively). Generally, synaptic complexes are assembled with two RSSs whose spacer lengths are different (the 12/23 rule). Subsequently, the RAG proteins catalyze a DNA double-strand break at each RSS (for reviews, see references 10 and 13), yielding a postcleavage complex containing four DNA ends: two blunt, 5Ј phosphorylated recombination signal ends and two coding ends terminating in DNA hairpin structures (41,42,46). The RAG proteins generate these recombination intermediates by nicking the DNA at the junction between the RSS and the coding sequence and then transferring the resulting 3Ј-OH to the opposing DNA strand by direct transesterification (32,...

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

“…5A). Consistent with our previous studies (27), the total abundance of reaction products generated after in vitro cleavage of an intact 23-RSS by WT and E649A RAGs is quite similar, but E649A RAGs more readily convert nicks to hairpin products, regardless of the presence of HMGB1. E649A RAGs also support higher levels of hairpin formation using a nicked 23-RSS substrate.…”

“…For in vitro cleavage experiments, the various plasmid substrates in Table 1 (100 ng; linearized with AatII or BglII) were incubated with the cMR1/cMR2 and HMGB1 proteins under the same reaction conditions as used to assess cleavage of oligonucleotide substrates. The cleaved DNA was ligated to linker DNA assembled from oligonucleotides DR19 and DR20 as described previously (27), and then the ligation reaction was terminated by incubation at 65°C for 30 min. Signal ends generated at the 12-RSS or 23-RSS positions were detected from one-tenth of the ligation reaction by PCR using recombinant TaqDNA polymerase (Invitrogen) and primer DR20 and either 12P (5Ј-TATTGTCTCATGAGCGGATAC-3Ј) or 23P (5Ј-GGTACATTGAGCAACTGACTG-3Ј), respectively.…”

“…Enhanced cRSS Cleavage by a Gain-of-function RAG1 Mutant-We recently identified an E-649A RAG1 mutant that exhibits increased cleavage activity relative to WT RAGs in the presence of Mg 2ϩ by promoting hairpin formation, possibly due to a defect in sensing 12/23-regulated synapsis (27). We hypothesized that this RAG1 mutant also supports enhanced cleavage of cRSS substrates.…”

RAG-Heptamer Interaction in the Synaptic Complex Is a Crucial Biochemical Checkpoint for the 12/23 Recombination Rule

“…1B). The HA3 phenotype is similar to that for the previously reported RAG1 mutant (E649A) that cleaves RSS DNA independent of the synapse formation (46). Two other mutants, HA1 and HA2, demonstrated elevated levels of hairpin formation by severalfold in the absence of the partner RSS, which could be further enhanced with the addition of partner RSS (Fig.…”

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

“…PCR products were fractionated on a 2% agarose gel and gel purified using the QIAQuick Gel Extraction kit (Qiagen Inc., Valencia, CA). Unlabeled DNA fragments of various lengths were similarly amplified by PCR from pJH299 or its derivative lacking the 23RSS (20) and purified using a QIAQuick PCR Cleanup kit.…”