The nonhomologous end-joining (NHEJ) pathway is responsible for rejoining the majority of double-strand breaks in mammalian cells, including the programmed breaks introduced by V(D)J recombination. The regulation of the enzymatic activities associated with this recombination pathway is still largely unknown. Here we report that human XRCC4 (for X-ray cross-complementation group 4), a protein essential for NHEJ, is subject to posttranslational protein modification. The modifier peptide, SUMO, can be added to XRCC4 both in vitro and in vivo. The site of modification is mapped to lysine 210 by using specific mutagenesis. A protein mutated such that it cannot be SUMOylated remains localized in the cytoplasm rather than accumulating in the nucleus. Cells expressing only the mutated protein are radiation sensitive and fail to complete V(D)J recombination. Genetic fusion of the SUMO sequence to the C terminus of the mutant restores nuclear localization and radiation resistance. The modification may serve a regulatory role. Our finding fits with an emerging literature associating SUMO modification with the control of the repair and recombination associated with DNA breaks. Double-strand DNA breaks (DSBs) arise naturally by means of a variety of mechanisms including direct breakage by ionizing radiation, replication of a nicked template, or enzymatic cleavage. Such events are likely to be lethal to a cell if left unresolved, so mechanisms to repair these lesions are quite important. The method that is used most commonly in mammalian cells is the nonhomologous end-joining (NHEJ) pathway (reviewed in references 24, 27, and 48). This pathway is of particular interest to immunologists because it is also essential for the completion of V(D)J recombination, the programmed DNA rearrangement that assembles the antigen receptors of B and T cells (reviewed in references 28 and 44).One of the indispensable proteins in the NHEJ pathway is XRCC4. This protein forms higher-order complexes with itself (35) and DNA ligase IV (5, 10) and is necessary for NHEJ activity in vivo. Since ligase IV alone is capable of joining DNA as a purified protein (42), the role of XRCC4 appears to be regulatory, perhaps through a structural contribution to the repair complex.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). Subsequently, other peptide modifiers have been found (more than 15 to date), the addition of which leads to diverse consequences for the target proteins. Among these modifiers are the SUMO proteins, whose name derives from the phrase "small ubiquitin-related modifier." The biochemistry and physiologic significance of this modification pathway have been recently reviewed (8, 13, 19, 36). SUMO modification is detected in several proteins conce...

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...

Ku70 is a protein that finds itself at the heart of several important cellular processes. It is essential to the Non-Homologous End Joining pathway as a part of the DNA-end binding complex, required for proper maintenance of telomeres and contributes to the DNA damage recognition and regulation of apoptosis. Forces that regulate Ku70 are therefore likely to have large consequences on the physiologic state of the cell. We report here that transient expression of the small protein SUMO resulted in a surprising increase in the abundance of Ku70. Surprisingly, the direct SUMOylation of Ku70 does not appear to be required for this effect. Rather, Ku70 appears to be stabilized through indirect effects on the rate of degradation. The same outcome was obtained by raising the expression of enzymes that promote SUMOylation. It is likely that many other proteins will be similarly regulated, providing a general control of cellular state.