The RAG1 N-terminal domain is an E3 ubiquitin ligase

Abstract

V(D)J recombination is a process through which DNA segments are cleaved, shuffled, and then religated to assemble functional immunoglobulin and T cell receptor loci. The process is required for the function of the adaptive immune response and provides a large degree of diversity needed by the antigen receptors. Recombination must also be highly regulated in terms of its DNA target specificity, developmental stage, and coordination with other nuclear events such as cell cycle. Undesired consequences of aberrant recombination include translocations like those associated with several leukemias (Bassing et al. 2002;Gellert 2002).The first enzymatic step in V(D)J recombination is the binding and then cleavage of the DNA substrate by a complex composed of the RAG1 and RAG2 proteins. These two proteins are the only known lymphoid-specific factors required for the reaction, and when expressed in nonlymphoid cells, they confer the ability to rearrange a test substrate (Oettinger et al. 1990). It came with some surprise to learn that the two proteins could be substantially truncated and yet remain functional in recombination (Sadofsky et al. 1993(Sadofsky et al. , 1994Silver et al. 1993;Cuomo and Oettinger 1994;Kirch et al. 1996). These smaller "core" portions of the RAG proteins have been easier to express and purify, allowing mechanistic studies of the cleavage reaction in vitro. These core proteins can participate in the formation of higher-order DNA-protein complexes, leading to synapsis and coupled cleavage of pairs of recombination targets (Bailin et al. 1999;Landree et al. 2001;Sadofsky 2001;Jones and Gellert 2002;Mundy et al. 2002;Swanson 2002).A linear representation of the RAG1 protein is shown as Figure 1A.After the cleavage step, the RAG proteins participate in the joining phase of the recombination reaction. This requires at least a structural contribution to binding the four DNA ends (Landree et al. 2001;Mo et al. 2001;Tsai et al. 2002) and may also reflect interactions with the DNA repair proteins needed for processing. Thus, the core region of RAG1 is apparently sufficient for all the DNA transactions in which the enzyme participates.Others have found that removal of the "dispensable" regions of the RAG proteins reduces the efficiency of the reaction (McMahan et al. 1997;Steen et al. 1999). In addition, the RAG proteins seem to have a role in lymphoid growth and regulation distinct from the nuclease function. Recombination of the heavy-chain locus in B cells typically involves D-to-J segment joining prior to recombination of the V segments. A mutant truncated in RAG2 is capable of catalyzing the first set of immunoglobulin rearrangements but not the later step (Kirch et al. 1998;Liang et al. 2002). It is also observed that a spontaneous frameshift mutation in RAG1 in a human patient with Omenn's syndrome leads to a more severe defect in B cell immunoglobulin rearrangement than at the T cell receptor locus (Santagata et al. 2000). These findings may reflect a role of the RAG proteins beyond their activity a...

SUMO Modification of Human XRCC4 Regulates Its Localization and Function in DNA Double-Strand Break Repair

“…Our interest in SUMOylation originated with the recognition that the E3 enzymes for this pathway frequently contain the RING motif. This protein structure is also common among the ubiquitin E3 enzymes and is found in the RAG1 protein important for V(D)J recombination (20,45,62). However, we do not have evidence of a functional link between this aspect of RAG1 and the SUMOylation of XRCC4 at this time.…”

“…Our previous finding of a ubiquitin ligase activity in RAG1 suggested that posttranslational peptide modifications may contribute to the regulation of V(D)J recombination (45,62). The recognition of covalent modification of a protein by the addition of a peptide modifier was first recognized for ubiquitylation (reviewed in references 39 and 60).…”

Ku70 is stabilized by increased cellular SUMO

“…Several other proteins involved in DNA repair or maintenance are also regulated by SUMO. Furthermore, the RING motif in RAG1 raises the possibility that ubiquitin or SUMO are involved in V(D)J recombination [12]. Here we report that raising the cellular level of SUMO or increasing the degree of protein SUMOylation stabilizes Ku70.…”

The NEF4 Complex Regulates Rad4 Levels and Utilizes Snf2/Swi2-Related ATPase Activity for Nucleotide Excision Repair

“…The RING domains of Rag1 and Rad5 are homologous to the Rad16 RING domain (Fig. 1A and B), and both Rag1 and Rad5 have E3 activity or are components of multisubunit E3 complexes (69,77). Thus, Rad16 is homologous to proteins with known E3 activity.…”