Patrick C. Swanson

V(D)J Recombination: Mechanisms of Initiation

V(D)J recombination assembles immunoglobulin and T cell receptor genes during lymphocyte development through a series of carefully orchestrated DNA breakage and rejoining events. DNA cleavage requires a series of protein-DNA complexes containing the RAG1 and RAG2 proteins and recombination signals that flank the recombining gene segments. In this review, we discuss recent advances in our understanding of the function and domain organization of the RAG proteins, the composition and structure of RAG-DNA complexes, and the pathways that lead to the formation of these complexes. We also consider the functional significance of RAG-mediated histone recognition and ubiquitin ligase activities, and the role played by RAG in ensuring proper repair of DNA breaks made during V(D)J recombination. Finally, we propose a model for the formation of RAG-DNA complexes that involves anchoring of RAG1 at the recombination signal nonamer and RAG2-dependent surveillance of adjoining DNA for suitable spacer and heptamer sequences.

A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex

The causes of spontaneous chromosomal translocations in somatic cells of biological organisms are largely unknown, although double-strand DNA breaks are required in all proposed mechanisms. The most common chromosomal abnormality in human cancer is the reciprocal translocation between chromosomes 14 and 18 (t(14;18)), which occurs in follicular lymphomas. The break at the immunoglobulin heavy-chain locus on chromosome 14 is an interruption of the normal V(D)J recombination process. But the breakage on chromosome 18, at the Bcl-2 gene, occurs within a confined 150-base-pair region (the major breakpoint region or Mbr) for reasons that have remained enigmatic. We have reproduced key features of the translocation process on an episome that propagates in human cells. The RAG complex—which is the normal enzyme for DNA cleavage at V, D or J segments—nicks the Bcl-2 Mbr in vitro and in vivo in a manner that reflects the pattern of the chromosomal translocations; however, the Mbr is not a V(D)J recombination signal. Rather the Bcl-2 Mbr assumes a non-B-form DNA structure within the chromosomes of human cells at 20–30% of alleles. Purified DNA assuming this structure contains stable regions of single-strandedness, which correspond well to the translocation regions in patients. Hence, a stable non-B-DNA structure in the human genome appears to be the basis for the fragility of the Bcl-2 Mbr, and the RAG complex is able to cleave this structure.

The bounty of RAGs: recombination signal complexes and reaction outcomes

Summary:  V(D)J recombination is a form of site‐specific DNA rearrangement through which antigen receptor genes are assembled. This process involves the breakage and reunion of DNA mediated by two lymphoid cell‐specific proteins, recombination activating genes RAG‐1 and RAG‐2, and ubiquitously expressed architectural DNA‐binding proteins and DNA‐repair factors. Here I review the progress toward understanding the composition, assembly, organization, and activity of the protein‐DNA complexes that support the initiation of V(D)J recombination, as well as the molecular basis for the sequence‐specific recognition of recombination signal sequences (RSSs) that are the targets of the RAG proteins. Parallels are drawn between V(D)J recombination and Tn5/Tn10 transposition with respect to the reactions, the proteins, and the protein‐DNA complexes involved in these processes. I also consider the relative roles of the different sequence elements within the RSS in recognition, cleavage, and post‐cleavage events. Finally, I discuss alternative DNA transactions mediated by the V(D)J recombinase, the protein‐DNA complexes that support them, and factors and forces that control them.

A RAG-1/RAG-2 tetramer supports 12/23-regulated synapsis, cleavage, and transposition of V(D)J recombination signals

ABSTRACT Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.

Double-Strand Break Formation by the RAG Complex at the Bcl-2 Major Breakpoint Region and at Other Non-B DNA Structures In Vitro

ABSTRACT The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.

Fine Structure and Activity of Discrete RAG-HMG Complexes on V(D)J Recombination Signals

ABSTRACT Two lymphoid cell-specific proteins, RAG-1 and RAG-2, initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences (RSSs). Although the RAG proteins themselves bind and cleave DNA substrates containing either a 12-RSS or a 23-RSS, DNA-bending proteins HMG-1 and HMG-2 are known to promote these processes, particularly with 23-RSS substrates. Using in-gel cleavage assays and DNA footprinting techniques, I analyzed the catalytic activity and protein-DNA contacts in discrete 12-RSS and 23-RSS complexes containing the RAG proteins and either HMG-1 or HMG-2. I found that both the cleavage activity and the pattern of protein-DNA contacts in RAG-HMG complexes assembled on 12-RSS substrates closely resembled those obtained from analogous 12-RSS complexes lacking HMG protein. In contrast, 23-RSS complexes containing both RAG proteins and either HMG-1 or HMG-2 exhibited enhanced cleavage activity and displayed an altered distribution of cleavage products compared to 23-RSS complexes containing only RAG-1 and RAG-2. Moreover, HMG-dependent heptamer contacts in 23-RSS complexes were observed. The protein-DNA contacts in RAG-RSS-HMG complexes assembled on 12-RSS or 23-RSS substrates were strikingly similar at comparable positions, suggesting that the RAG proteins mediate HMG-dependent heptamer contacts in 23-RSS complexes. Results of ethylation interference experiments suggest that the HMG protein is positioned 5′ of the nonamer in 23-RSS complexes, interacting largely with the side of the duplex opposite the one contacting the RAG proteins. Thus, HMG protein plays the dual role of bringing critical elements of the 23-RSS heptamer into the same phase as the 12-RSS to promote RAG binding and assisting in the catalysis of 23-RSS cleavage.

The DDE motif in RAG-1 is contributed in trans to a single active site that catalyzes the nicking and transesterification steps of V(D)J recombination.

ABSTRACT The process of assembling immunoglobulin and T-cell receptor genes from variable (V), diversity (D), and joining (J) gene segments, called V(D)J recombination, involves the introduction of DNA breaks at recombination signals. DNA cleavage is catalyzed by RAG-1 and RAG-2 in two chemical steps: first-strand nicking, followed by hairpin formation via direct transesterification. In vitro, these reactions minimally proceed in discrete protein-DNA complexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a single recombination signal sequence. Recently, a DDE triad of carboxylate residues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral integrase active sites. To investigate which RAG-1 subunit contributes the residues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing RAG-2 and a RAG-1 heterodimer that carried an active-site mutation targeted to the same or opposite RAG-1 subunit mutated to be incompetent for DNA binding. The results show that the DDE triad is contributed to a single recombinase active site, which catalyzes the nicking and transesterification steps of V(D)J recombination by a single RAG-1 subunit opposite the one bound to the nonamer of the recombination signal undergoing cleavage (cleavage intrans). The implications of a trans cleavage mode observed in these complexes on the organization of the V(D)J synaptic complex are discussed.

V(D)J recombinase binding and cleavage of cryptic recombination signal sequences identified from lymphoid malignancies

V(D)J recombination is a process integral to lymphocyte development. However, this process is not always benign, since certain lymphoid malignancies exhibit recurrent chromosomal abnormalities, such as translocations and deletions, that harbor molecular signatures suggesting an origin from aberrant V(D)J recombination. Translocations involving LMO2, TAL1, Ttg-1, and Hox11, as well as a recurrent interstitial deletion at 1p32 involving SIL/SCL, are cited examples of illegitimate V(D)J recombination. Previous studies using extrachromosomal substrates reveal that cryptic recombination signal sequences (cRSSs) identified near the translocation breakpoint in these examples support V(D)J recombination with efficiencies ranging from about 30- to 20,000-fold less than bona fide V(D)J recombination signals. To understand the molecular basis for these large differences, we investigated the binding and cleavage of these cRSSs by the RAG1/2 proteins that initiate V(D)J recombination. We find that the RAG proteins comparably bind all cRSSs tested, albeit more poorly than a consensus RSS. We show that four cRSSs that support levels of V(D)J recombination above background levels in cell culture (LMO2, TAL1, Ttg-1, and SIL) are also cleaved by the RAG proteins in vitro with efficiencies ranging from 18 to 70% of a consensus RSS. Cleavage of LMO2 and Ttg-1 by the RAG proteins can also be detected in cell culture using ligation-mediated PCR. In contrast, Hox11 and SCL are nicked but not cleaved efficiently in vitro, and cleavage at other adventitious sites in plasmid substrates may also limit the ability to detect recombination activity at these cRSSs in cell culture.

Evidence for Ku70/Ku80 association with full-length RAG1

Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.

RAG and HMGB1 Proteins: Purification and Biochemical Analysis of Recombination Signal Complexes

Two lymphoid cell-specific proteins, called RAG-1 and RAG-2, initiate the process of antigen receptor gene rearrangement, termed V(D)J recombination, by assembling a protein-DNA complex with two recombination signal sequences (RSSs), each of which adjoins a different receptor gene segment, and then introducing a DNA double strand break at the end of each RSS. The study of RAG-RSS complex assembly and activity has been facilitated by the development of methods to purify the RAG proteins and members of the HMG-box family of high mobility group proteins such as HMGB1 that promote RAG binding and cleavage activity in vitro. This chapter describes the purification of recombinant truncated and full-length RAG-1 and RAG-2 expressed transiently in mammalian cells, as well as the purification of bacterially expressed full-length HMGB1. In addition, it details several experimental procedures used in our laboratory to study RAG-RSS complex formation and function in vitro.