David Brian Roth, M.D., Ph.D.

The RAG2 C terminus suppresses genomic instability and lymphomagenesis
Deriano, Ludovic; Chaumeil, Julie; Coussens, Marc; Multani, Asha; Chou, YiFan; Alekseyenko, Alexander V; Chang, Sandy; Skok, Jane A; Roth, David B
2011 Mar 3;471(7336):119-123, Nature
Misrepair of DNA double-strand breaks produced by the V(D)J recombinase (the RAG1/RAG2 proteins) at immunoglobulin (Ig) and T cell receptor (Tcr) loci has been implicated in pathogenesis of lymphoid malignancies in humans and in mice. Defects in DNA damage response factors such as ataxia telangiectasia mutated (ATM) protein and combined deficiencies in classical non-homologous end joining and p53 predispose to RAG-initiated genomic rearrangements and lymphomagenesis. Although we showed previously that RAG1/RAG2 shepherd the broken DNA ends to classical non-homologous end joining for proper repair, roles for the RAG proteins in preserving genomic stability remain poorly defined. Here we show that the RAG2 carboxy (C) terminus, although dispensable for recombination, is critical for maintaining genomic stability. Thymocytes from 'core' Rag2 homozygotes (Rag2(c/c) mice) show dramatic disruption of Tcralpha/delta locus integrity. Furthermore, all Rag2(c/c) p53(-/-) mice, unlike Rag1(c/c) p53(-/-) and p53(-/-) animals, rapidly develop thymic lymphomas bearing complex chromosomal translocations, amplifications and deletions involving the Tcralpha/delta and Igh loci. We also find these features in lymphomas from Atm(-/-) mice. We show that, like ATM-deficiency, core RAG2 severely destabilizes the RAG post-cleavage complex. These results reveal a novel genome guardian role for RAG2 and suggest that similar 'end release/end persistence' mechanisms underlie genomic instability and lymphomagenesis in Rag2(c/c) p53(-/-) and Atm(-/-) mice
— id: 128877, year: 2011, vol: 471, page: 119, stat: Journal Article,

Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
Arnal, Suzzette M; Holub, Abigail J; Salus, Sandra S; Roth, David B
2010 May;38(9):2944-2954, Nucleic acids research
V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC's ability to restrict repair pathway choice
— id: 109790, year: 2010, vol: 38, page: 2944, stat: Journal Article,

Base flipping in V(D)J recombination: insights into the mechanism of hairpin formation, the 12/23 rule, and the coordination of double-strand breaks
Bischerour, Julien; Lu, Catherine; Roth, David B; Chalmers, Ronald
2009 Nov;29(21):5889-5899, Molecular & cellular biology
Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position -1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex
— id: 115414, year: 2009, vol: 29, page: 5889, stat: Journal Article,

Recent insights into the formation of RAG-induced chromosomal translocations
Brandt, Vicky L; Roth, David B
2009 ;650:32-45, Advances in experimental medicine & biology
Chromosomal translocations are found in many types of tumors, where they may be either a cause or a result of malignant transformation. In lymphoid neoplasms, however, it is dear that pathogenesis is initiated by any of a number of recurrent DNA rearrangements. These particular translocations typically place an oncogene under the regulatory control of an Ig or TCR gene promoter, dysregulating cell growth, differentiation, or apoptosis. Given that physiological DNA rearrangements (V(D)J and class switch recombination) are integral to lymphocyte development, it is critical to understand how genomic stability is maintained during these processes. Recent advances in our understanding of DNA damage signaling and repair have provided clues to the kinds of mechanisms that lead to V(D)J-mediated translocations. In turn, investigations into the regulation of V(D)J joining have illuminated a formerly obscure pathway of DNA repair known as alternative NHEJ, which is error-prone and frequently involved in translocations. In this chapter we consider recent advances in our understanding of the functions of the RAG proteins, RAG interactions with DNA repair pathways, damage signaling and chromosome biology, all of which shed light on how mistakes at different stages of V(D)J recombination might lead to leukemias and lymphomas
— id: 102159, year: 2009, vol: 650, page: 32, stat: Journal Article,

Roles for NBS1 in alternative nonhomologous end-joining of V(D)J recombination intermediates
Deriano, Ludovic; Stracker, Travis H; Baker, Annalee; Petrini, John H J; Roth, David B
2009 Apr 10;34(1):13-25, Molecular cell
Recent work has highlighted the importance of alternative, error-prone mechanisms for joining DNA double-strand breaks (DSBs) in mammalian cells. These noncanonical, nonhomologous end-joining (NHEJ) pathways threaten genomic stability but remain poorly characterized. The RAG postcleavage complex normally prevents V(D)J recombination-associated DSBs from accessing alternative NHEJ. Because the MRE11/RAD50/NBS1 complex localizes to RAG-mediated DSBs and possesses DNA end tethering, processing, and joining activities, we asked whether it plays a role in the mechanism of alternative NHEJ or participates in regulating access of DSBs to alternative repair pathways. We find that NBS1 is required for alternative NHEJ of hairpin coding ends, suppresses alternative NHEJ of signal ends, and promotes proper resolution of inversional recombination intermediates. These data demonstrate that the MRE11 complex functions at two distinct levels, regulating repair pathway choice (likely through enhancing the stability of DNA end complexes) and participating in alternative NHEJ of coding ends
— id: 100512, year: 2009, vol: 34, page: 13, stat: Journal Article,

Men'ekigaku irasutoreiteddo = [Immunology]
Male, David; Brostoff, Jonathan; Roth, David B; Roitt, Ivan Maurice; Takatsu, Kiyoshi; Kiyono, Hiroshi; Miyake, Kensuke
Tokyo : Nankodo, 2009,
— id: 2214, year: 2009, vol: , page: , stat: ,

Artemis and nonhomologous end joining-independent influence of DNA-dependent protein kinase catalytic subunit on chromosome stability
Stracker, Travis H; Williams, Bret R; Deriano, Ludovic; Theunissen, Jan W; Adelman, Carrie A; Roth, David B; Petrini, John H J
2009 Jan;29(2):503-514, Molecular & cellular biology
Deficiency in both ATM and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is synthetically lethal in developing mouse embryos. Using mice that phenocopy diverse aspects of Atm deficiency, we have analyzed the genetic requirements for embryonic lethality in the absence of functional DNA-PKcs. Similar to the loss of ATM, hypomorphic mutations of Mre11 (Mre11(ATLD1)) led to synthetic lethality when juxtaposed with DNA-PKcs deficiency (Prkdc(scid)). In contrast, the more moderate DNA double-strand break response defects associated with the Nbs1(DeltaB) allele permitted viability of some Nbs1(DeltaB/DeltaB) Prkdc(scid/scid) embryos. Cell cultures from Nbs1(DeltaB/DeltaB) Prkdc(scid/scid) embryos displayed severe defects, including premature senescence, mitotic aberrations, sensitivity to ionizing radiation, altered checkpoint responses, and increased chromosome instability. The known functions of DNA-PKcs in the regulation of Artemis nuclease activity or nonhomologous end joining-mediated repair do not appear to underlie the severe genetic interaction. Our results reveal a role for DNA-PKcs in the maintenance of S/G(2)-phase chromosome stability and in the induction of cell cycle checkpoint responses
— id: 115415, year: 2009, vol: 29, page: 503, stat: Journal Article,

G.O.D.'s Holy Grail: discovery of the RAG proteins
Brandt, Vicky L; Roth, David B
2008 Jan 1;180(1):3-4, Journal of immunology
— id: 76338, year: 2008, vol: 180, page: 3, stat: Journal Article,

Understanding and re-engineering nucleoprotein machines to cure human disease
Dynan, William; Takeda, Yoshihiko; Roth, David; Bao, Gang
2008 Feb;3(1):93-105, Nanomedicine
The mammalian nucleus is filled with self-organizing, nanometer-scale nucleoprotein machines that carry out DNA replication, RNA biogenesis and DNA repair. We discuss, as a model, the nonhomologous end-joining (NHEJ) machine, which repairs DNA double-strand breaks. The NHEJ machine consists of six core polypeptides and 10-20 ancillary polypeptides. A full understanding of its design principles will require measuring the behavior of single NHEJ complexes in living cells, using a Nano Toolbox that includes bright, stable, biocompatible fluorophores, efficient protein and nucleic acid-tagging strategies, and sensitive, high-resolution imaging methods. Taking inspiration from natural examples, it might be possible to adapt and redesign the NHEJ machine to precisely correct mutations responsible for common human diseases, such as K-ras in lung cancer or human papillomavirus E6 and E7 genes in cervical and oral cancers
— id: 141141, year: 2008, vol: 3, page: 93, stat: Journal Article,

Understanding how the V(D)J recombinase catalyzes transesterification: distinctions between DNA cleavage and transposition
Lu, Catherine P; Posey, Jennifer E; Roth, David B
2008 May;36(9):2864-2873, Nucleic acids research
The Rag1 and Rag2 proteins initiate V(D)J recombination by introducing site-specific DNA double-strand breaks. Cleavage occurs by nicking one DNA strand, followed by a one-step transesterification reaction that forms a DNA hairpin structure. A similar reaction allows Rag transposition, in which the 3'-OH groups produced by Rag cleavage are joined to target DNA. The Rag1 active site DDE triad clearly plays a catalytic role in both cleavage and transposition, but no other residues in Rag1 responsible for transesterification have been identified. Furthermore, although Rag2 is essential for both cleavage and transposition, the nature of its involvement is unknown. Here, we identify basic amino acids in the catalytic core of Rag1 specifically important for transesterification. We also show that some Rag1 mutants with severe defects in hairpin formation nonetheless catalyze substantial levels of transposition. Lastly, we show that a catalytically defective Rag2 mutant is impaired in target capture and displays a novel form of coding flank sensitivity. These findings provide the first identification of components of Rag1 that are specifically required for transesterification and suggest an unexpected role for Rag2 in DNA cleavage and transposition
— id: 79300, year: 2008, vol: 36, page: 2864, stat: Journal Article,

A RAG1 mutation found in Omenn syndrome causes coding flank hypersensitivity: a novel mechanism for antigen receptor repertoire restriction
Wong, Serre-Yu; Lu, Catherine P; Roth, David B
2008 Sep 15;181(6):4124-4130, Journal of immunology
Hypomorphic RAG mutants with severely reduced V(D)J recombination activity cause Omenn Syndrome (OS), an immunodeficiency with features of immune dysregulation and a restricted TCR repertoire. Precisely how RAG mutants produce autoimmune and allergic symptoms has been unclear. Current models posit that the severe recombination defect restricts the number of lymphocyte clones, a few of which are selected upon Ag exposure. We show that murine RAG1 R972Q, corresponding to an OS mutation, renders the recombinase hypersensitive to selected coding sequences at the hairpin formation step. Other RAG1 OS mutants tested do not manifest this sequence sensitivity. These new data support a novel mechanism for OS: by selectively impairing recombination at certain coding flanks, a RAG mutant can cause primary repertoire restriction, as opposed to a more random, limited repertoire that develops secondary to severely diminished recombination activity
— id: 93364, year: 2008, vol: 181, page: 4124, stat: Journal Article,

Excised V(D)J recombination byproducts threaten genomic integrity
Arnal, Suzzette M; Roth, David B
2007 Jul;28(7):289-292, Trends in immunology
Signal joints were long considered to be inert byproducts of V(D)J recombination that protect the genome from illegitimate rearrangements. However, increasing evidence suggests that signal joints are not inert and could pose a threat to genomic stability. A recent study from Nadel and colleagues shows that episomal signal joints readily undergo trans recombination, resulting in their insertion into chromosomal DNA
— id: 73949, year: 2007, vol: 28, page: 289, stat: Journal Article,

Rag mutations reveal robust alternative end joining
Corneo, Barbara; Wendland, Rebecca L; Deriano, Ludovic; Cui, Xiaoping; Klein, Isaac A; Wong, Serre-Yu; Arnal, Suzzette; Holub, Abigail J; Weller, Geoffrey R; Pancake, Bette A; Shah, Sundeep; Brandt, Vicky L; Meek, Katheryn; Roth, David B
2007 Sep 27;449(7161):483-486, Nature
Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1-Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, 'alternative NHEJ', appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell's overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination
— id: 74579, year: 2007, vol: 449, page: 483, stat: Journal Article,

Murine models of Omenn syndrome
Wong, Serre-Yu; Roth, David B
2007 May;117(5):1213-1216, Journal of clinical investigation
In the 40 years since Harvard medical student Gilbert Omenn first described a rare, inherited disorder producing a paradoxical combination of immunodeficiency and immune dysregulation, the pathogenesis of Omenn syndrome (OS) has remained mysterious. In separate studies reported in this issue of the JCI, two mouse models bearing mutations in the V(D)J recombinase analogous to those causing human OS have been shown to recapitulate the disease and provide insight into the genesis of immunodeficiency combined with autoimmunity and atopy in OS and other disease settings (see the related articles beginning on pages 1260 and 1270).
— id: 72873, year: 2007, vol: 117, page: 1213, stat: Journal Article,

Amino acid residues in Rag1 crucial for DNA hairpin formation
Lu, Catherine P; Sandoval, Hector; Brandt, Vicky L; Rice, Phoebe A; Roth, David B
2006 Nov;13(11):1010-1015, Nature structural & molecular biology
The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases
— id: 69318, year: 2006, vol: 13, page: 1010, stat: Journal Article,

Immunology
Male, David K; Brostoff, Jonathan; Roth, David B; Roitt, Ivan Maurice
Edinburgh : Elsevier Mosby, 2006,
— id: 2215, year: 2006, vol: , page: , stat: ,

Target DNA structure plays a critical role in RAG transposition
Posey, Jennifer E; Pytlos, Malgorzata J; Sinden, Richard R; Roth, David B
2006 Nov;4(11):e350-e350, PLoS biology
Antigen receptor gene rearrangements are initiated by the RAG1/2 protein complex, which recognizes specific DNA sequences termed RSS (recombination signal sequences). The RAG recombinase can also catalyze transposition: integration of a DNA segment bounded by RSS into an unrelated DNA target. For reasons that remain poorly understood, such events occur readily in vitro, but are rarely detected in vivo. Previous work showed that non-B DNA structures, particularly hairpins, stimulate transposition. Here we show that the sequence of the four nucleotides at a hairpin tip modulates transposition efficiency over a surprisingly wide (>100-fold) range. Some hairpin targets stimulate extraordinarily efficient transposition (up to 15%); one serves as a potent and specific transposition inhibitor, blocking capture of targets and destabilizing preformed target capture complexes. These findings suggest novel regulatory possibilities and may provide insight into the activities of other transposases
— id: 69317, year: 2006, vol: 4, page: e350, stat: Journal Article,

V(D)J recombination: how to tame a transposase
Brandt, Vicky L; Roth, David B
2004 Aug;200:249-260, Immunological reviews
Since the discovery that the recombination-activating gene (RAG) proteins were capable of transposition in vitro, investigators have been trying to uncover instances of transposition in vivo and understand how this transposase has been harnessed to do useful work while being inhibited from causing deleterious chromosome rearrangements. How to preserve the capacity of the recombinase to promote a certain class of rearrangements while curtailing its ability to catalyze others is an interesting problem. In this review, we examine the progress that has been made toward understanding the regulatory mechanisms that prohibit transposition in order to formulate a model that takes into account the diverse observations that have been made over the last 15 years. First, we touch on the striking mechanistic similarities between transposition and V(D)J recombination and review evidence suggesting that the RAG proteins may be members of the retroviral integrase superfamily. We then dispense with an old theory that certain standard products of V(D)J recombination called signal joints protect against deleterious transposition events. Finally, we discuss the evidence that target capture could serve a regulatory role and close with an analysis of hairpins as preferred targets for RAG-mediated transposition. These novel strategies for harnessing the RAG transposase not only shed light on V(D)J recombination but also may provide insight into the regulation of other transposases
— id: 45018, year: 2004, vol: 200, page: 249, stat: Journal Article,

B cell development leads off with a base hit: dU:dG mismatches in class switching and hypermutation
Lee, Gregory S; Brandt, Vicky L; Roth, David B
2004 Nov 19;16(4):505-508, Molecular cell
The mechanisms underlying somatic hypermutation (SHM) and class switch recombination (CSR) have been the subject of much debate. Recent studies from the Neuberger and Honjo labs have lent insight into these distinct processes, and we discuss a new, comprehensive model for how AID, uracil DNA glycosylase (UNG) and the mismatch repair system function in both SHM and CSR
— id: 47773, year: 2004, vol: 16, page: 505, stat: Journal Article,

RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination
Lee, Gregory S; Neiditch, Matthew B; Salus, Sandra S; Roth, David B
2004 Apr 16;117(2):171-184, Cell
The two major pathways for repairing double-strand breaks (DSBs), homologous recombination and nonhomologous end joining (NHEJ), have traditionally been thought to operate in different stages of the cell cycle. This division of labor is not absolute, however, and precisely what governs the choice of pathway to repair a given DSB has remained enigmatic. We pursued this question by studying the site-specific DSBs created during V(D)J recombination, which relies on classical NHEJ to repair the broken ends. We show that mutations that form unstable RAG postcleavage complexes allow DNA ends to participate in both homologous recombination and the error-prone alternative NHEJ pathway. By abrogating a key function of the complex, these mutations reveal it to be a molecular shepherd that guides DSBs to the proper pathway. We also find that RAG-mediated nicks efficiently stimulate homologous recombination and discuss the implications of these findings for oncogenic chromosomal rearrangements, evolution, and gene targeting
— id: 45020, year: 2004, vol: 117, page: 171, stat: Journal Article,

Paradigm switching in the germinal center
Posey, Jennifer E; Brandt, Vicky L; Roth, David B
2004 May;5(5):476-477, Nature immunology
— id: 45019, year: 2004, vol: 5, page: 476, stat: Journal Article,

Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks
Sandor, Zoltan; Calicchio, Monica L; Sargent, R Geoffrey; Roth, David B; Wilson, John H
2004 ;32(6):1866-1873, Nucleic acids research
Loss or addition of nucleotides at junctions generated by V(D)J recombination significantly expands the antigen-receptor repertoire. Addition of nontemplated (N) nucleotides is carried out by terminal deoxynucleotidyl transferase (TdT), whose only known physiological role is to create diversity at V(D)J junctions during lymphocyte development. Although purified TdT can act at free DNA ends, its ability to add nucleotides (i.e. form N regions) at coding joints appears to depend on the nonhomologous end-joining factor Ku80. Because the DNA ends generated during V(D)J rearrangements remain associated with the RAG proteins after cleavage, TdT might be targeted for N region addition through interactions with RAG proteins or with Ku80 during remodeling of the post-cleavage complex. Such regulated access would help to prevent TdT from acting at other types of broken ends and degrading the fidelity of end joining. To test this hypothesis, we measured TdT's ability to add nucleotides to endonuclease-induced chromosomal and extrachromosomal breaks. In both cases TdT added nucleotides efficiently to the cleaved DNA ends. Strikingly, the frequency of N regions at non-V(D)J-generated ends was not dependent on Ku80. Thus our results suggest that Ku80 is required to allow TdT access to RAG post-cleavage complexes, providing support for the hypothesis that Ku is involved in disassembling or remodeling the post-cleavage complex. We also found that N regions were abnormally long in the absence of Ku80, indicating that Ku80 may regulate TdT's activity at DNA ends in vivo
— id: 45021, year: 2004, vol: 32, page: 1866, stat: Journal Article,

Doing more with less in bacterial DNA repair
Weller, Geoffrey R; Brandt, Vicky L; Roth, David B
2004 Dec;11(12):1158-1159, Nature structural & molecular biology
— id: 115416, year: 2004, vol: 11, page: 1158, stat: Journal Article,

Artemis: guarding small children and, now, the genome
Brandt, Vicky L; Roth, David B
2003 Feb;111(3):315-316, Journal of clinical investigation
— id: 37381, year: 2003, vol: 111, page: 315, stat: Journal Article,

Restraining the V(D)J recombinase
Roth, David B
2003 Aug;3(8):656-666, Nature reviews. Immunology
Chromosome breakage--a dangerous event that has triggered the evolution of several double-strand break repair pathways--has been co-opted by the immune system as an integral part of B- and T-cell development. This is a daring strategy, as improper repair can be deadly for the cell, if not for the whole organism. Even more daring, however, is the choice of a promiscuous transposase as the nuclease responsible for chromosome breakage, as the possibility of transposition brings an entirely new set of risks. What mechanisms constrain the dangerous potential of the recombinase and preserve genomic integrity during immune-system development?
— id: 39069, year: 2003, vol: 3, page: 656, stat: Journal Article,

Contribution of Vh gene replacement to the primary B cell repertoire
Zhang, Zhixin; Zemlin, Michael; Wang, Yui-Hsi; Munfus, Delicia; Huye, Leslie E; Findley, Harry W; Bridges, S Louis; Roth, David B; Burrows, Peter D; Cooper, Max D
2003 Jul;19(1):21-31, Immunity
V(H) replacement has been proposed as one way to modify unwanted antibody specificities, but analysis of this mechanism has been limited without a dynamic cellular model. We describe a human cell line that spontaneously undergoes serial V(H) gene replacement mediated by cryptic recombination signal sequences (cRSS) located near the 3' end of V(H) genes. Recombination-activating gene products, RAG-1 and RAG-2, bind and cleave the cRSS to generate DNA deletion circles during the V(H) replacement process. A V(H) replacement contribution to normal repertoire development is revealed by the identification of V(H) replacement 'footprints' in IgH sequences and double-stranded DNA breaks at V(H) cRSS sites in immature B cells. Surprisingly, the residual 3' sequences of replaced V(H) genes contribute charged amino acids to the CDR3 region, a hallmark of autoreactive antibodies
— id: 37380, year: 2003, vol: 19, page: 21, stat: Journal Article,

Cancer predisposition and hematopoietic failure in Rad50(S/S) mice
Bender, Carla F; Sikes, Michael L; Sullivan, Ruth; Huye, Leslie Erskine; Le Beau, Michelle M; Roth, David B; Mirzoeva, Olga K; Oltz, Eugene M; Petrini, John H J
2002 Sep 1;16(17):2237-2251, Genes & development
Mre11, Rad50, and Nbs1 function in a protein complex that is central to the metabolism of chromosome breaks. Null mutants of each are inviable. We demonstrate here that hypomorphic Rad50 mutant mice (Rad50(S/S) mice) exhibited growth defects and cancer predisposition. Rad50(S/S) mice died with complete bone marrow depletion as a result of progressive hematopoietic stem cell failure. Similar attrition occurred in spermatogenic cells. In both contexts, attrition was substantially mitigated by p53 deficiency, whereas the tumor latency of p53(-/-) and p53(+/-) animals was reduced by Rad50(S/S). Indices of genotoxic stress and chromosomal rearrangements were evident in Rad50(S/S) cultured cells, as well as in Rad50(S/S) and p53(-/-) Rad50(S/S) lymphomas, suggesting that the Rad50(S/S) phenotype was attributable to chromosomal instability. These outcomes were not associated with overt defects in the Mre11 complex's previously established double strand break repair and cell cycle checkpoint regulation functions. The data indicate that even subtle perturbation of Mre11 complex functions results in severe genotoxic stress, and that the complex is critically important for homeostasis of proliferative tissues
— id: 37382, year: 2002, vol: 16, page: 2237, stat: Journal Article,

A recombinase diversified: new functions of the RAG proteins
Brandt, Vicky L; Roth, David B
2002 Apr;14(2):224-229, Current opinion in immunology
The RAG proteins were long thought to serve merely as a nuclease, initiating recombination by cleaving DNA. Recent work has shown, however, that these proteins are essential for many steps in the recombination pathway, such as opening hairpins and joining broken DNA ends, and that they can also act as a transposase, targeting distorted DNA structures such as hairpins
— id: 37388, year: 2002, vol: 14, page: 224, stat: Journal Article,

Mutational analysis of all conserved basic amino acids in RAG-1 reveals catalytic, step arrest, and joining-deficient mutants in the V(D)J recombinase
Huye, Leslie E; Purugganan, Mary M; Jiang, Ming-Ming; Roth, David B
2002 May;22(10):3460-3473, Molecular & cellular biology
Although both RAG-1 and RAG-2 are required for all steps of V(D)J recombination, little is known about the specific contribution of either protein to these steps. RAG-1 contains three acidic active-site amino acids that are thought to coordinate catalytic metal ions. To search for additional catalytic amino acids and to better define the functional anatomy of RAG-1, we mutated all 86 conserved basic amino acids to alanine and evaluated the mutant proteins for DNA binding, nicking, hairpin formation, and joining. We found several amino acids outside of the canonical nonamer-binding domain that are critical for DNA binding, several step arrest mutants with defects in nicking or hairpin formation, and four RAG-1 mutants defective specifically for joining. Analysis of coding joints formed by some of these mutants revealed excessive deletions, frequent use of short sequence homologies, and unusually long palindromic junctional inserts, known as P nucleotides, that result from aberrant hairpin opening. These features characterize junctions found in scid mice, which are deficient for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), suggesting that the RAG proteins and DNA-PKcs perform overlapping functions in coding joint formation. Interestingly, the amino acids that are altered in 12 of our mutants are also mutated in human inherited immunodeficiency syndromes. Our analysis of these mutants provides insights into the molecular mechanisms underlying these disorders
— id: 37385, year: 2002, vol: 22, page: 3460, stat: Journal Article,

Targeted transposition by the V(D)J recombinase
Lee, Gregory S; Neiditch, Matthew B; Sinden, Richard R; Roth, David B
2002 Apr;22(7):2068-2077, Molecular & cellular biology
Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by--and specifically targeted to--hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected
— id: 37387, year: 2002, vol: 22, page: 2068, stat: Journal Article,

The V(D)J recombinase efficiently cleaves and transposes signal joints
Neiditch, Matthew B; Lee, Gregory S; Huye, Leslie E; Brandt, Vicky L; Roth, David B
2002 Apr;9(4):871-878, Molecular cell
V(D)J recombination generates two types of products: coding joints, which constitute the rearranged variable regions of antigen receptor genes, and signal joints, which often form on immunologically irrelevant, excised circular molecules that are lost during cell division. It has been widely believed that signal joints simply convert reactive broken DNA ends into safe, inert products. Yet two curious in vivo observations made us question this assumption: signal ends are far more abundant than coding ends, and signal joints form only after RAG expression is downregulated. In fact, we find that signal joints are not at all inert; they are cleaved quite efficiently in vivo and in vitro by a nick-nick mechanism and form an excellent substrate for RAG-mediated transposition in vitro, possibly explaining how genomic stability in lymphocytes may be compromised
— id: 37384, year: 2002, vol: 9, page: 871, stat: Journal Article,

Amplifying mechanisms of lymphomagenesis
Roth, David B
2002 Jul;10(1):1-2, Molecular cell
Mice doubly deficient for either XRCC4 or DNA ligase IV and p53 invariably develop lymphomas bearing characteristic chromosome translocations with gene amplification. A recent study highlights the importance of nonclassical end joining mechanisms in the formation of these oncogenic DNA rearrangements
— id: 37383, year: 2002, vol: 10, page: 1, stat: Journal Article,

Distinct and opposite diversifying activities of terminal transferase splice variants
Thai, To-Ha; Purugganan, Mary M; Roth, David B; Kearney, John F
2002 May;3(5):457-462, Nature immunology
The short splice variant of mouse terminal deoxynucleotidyl transferase (TdTS) catalyzes the addition of nontemplated nucleotides (N addition) at the coding joins of B cell and T cell antigen receptor genes. However, the activity and function of the long isoform of TdT (TdTL) have not been determined. We show here, in vitro and in vivo, that TdTL is a 3'-->5' exonuclease that catalyzes the deletion of nucleotides at coding joins. These findings suggest that the two TdT isoforms may act in concert to preserve the integrity of the variable region of antigen receptors while generating diversity
— id: 37386, year: 2002, vol: 3, page: 457, stat: Journal Article,

Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells
d'Adda di Fagagna, F; Hande, M P; Tong, W M; Roth, D; Lansdorp, P M; Wang, Z Q; Jackson, S P
2001 Aug 7;11(15):1192-1196, Current biology. CB
DNA repair by nonhomologous end-joining (NHEJ) relies on the Ku70:Ku80 heterodimer in species ranging from yeast to man. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, Ku also controls telomere functions. Here, we show that Ku70, Ku80, and DNA-PKcs, with which Ku interacts, associate in vivo with telomeric DNA in several human cell types, and we show that these associations are not significantly affected by DNA-damaging agents. We also demonstrate that inactivation of Ku80 or Ku70 in the mouse yields telomeric shortening in various primary cell types at different developmental stages. By contrast, telomere length is not altered in cells impaired in XRCC4 or DNA ligase IV, two other NHEJ components. We also observe higher genomic instability in Ku-deficient cells than in XRCC4-null cells. This suggests that chromosomal instability of Ku-deficient cells results from a combination of compromised telomere stability and defective NHEJ
— id: 111487, year: 2001, vol: 11, page: 1192, stat: Journal Article,

Conditional RAG-1 mutants block the hairpin formation step of V(D)J recombination
Kale SB; Landree MA; Roth DB
2001 Jan;21(2):459-466, Molecular & cellular biology
Hairpin formation serves an important regulatory role in V(D)J recombination because it requires synapsis of an appropriate pair of recombination sites. How hairpin formation is regulated and which regions of the RAG proteins perform this step remain unknown. We analyzed two conditional RAG-1 mutants that affect residues quite close in the primary sequence to an active site amino acid (D600), and we found that they exhibit severely impaired recombination in the presence of certain cleavage site sequences. These mutants are specifically defective for the formation of hairpins, providing the first identification of a region of the V(D)J recombinase necessary for this reaction. Substrates containing mismatched bases at the cleavage site rescued hairpin formation by both mutants, which suggests that the mutations affect the generation of a distorted or unwound DNA intermediate that has been implicated in hairpin formation. Our results also indicate that this region of RAG-1 may be important for coupling hairpin formation to synapsis
— id: 37396, year: 2001, vol: 21, page: 459, stat: Journal Article,

Functional organization of single and paired V(D)J cleavage complexes
Landree MA; Kale SB; Roth DB
2001 Jul;21(13):4256-4264, Molecular & cellular biology
RAG-1 and RAG-2 initiate V(D)J recombination by binding to specific recognition sequences (RSS) and then cleave the DNA in two steps: nicking and hairpin formation. Recent work has established that a dimer of RAG-1 and either one or two monomers of RAG-2 bind to a single RSS, but the enzymatic contributions of the RAG molecules within this nucleoprotein complex and its functional organization have not been elucidated. Using heterodimeric protein preparations containing both wild-type and catalytically deficient RAG-1 molecules, we found that one active monomer is sufficient for both nicking and hairpin formation at a single RSS, demonstrating that a single active site can carry out both cleavage steps. Furthermore, the mutant heterodimers efficiently cleaved both RSS in a synaptic complex. These results strongly suggest that two RAG-1 dimers are responsible for RSS cleavage in a synaptic complex, with one monomer of each dimer catalyzing both nicking and hairpin formation at each RSS
— id: 37390, year: 2001, vol: 21, page: 4256, stat: Journal Article,

RAG transposase can capture and commit to target DNA before or after donor cleavage
Neiditch MB; Lee GS; Landree MA; Roth DB
2001 Jul;21(13):4302-4310, Molecular & cellular biology
The discovery that the V(D)J recombinase functions as a transposase in vitro suggests that transposition by this system might be a potent source of genomic instability. To gain insight into the mechanisms that regulate transposition, we investigated a phenomenon termed target commitment that reflects a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productive complexes with target DNA both before and after donor cleavage, and our data indicate that the rate-limiting step for transposition occurs after target capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may result in a preference for transposition into nearby targets, such as immunoglobulin and T-cell receptor loci. This could bias transposition toward relatively 'safe' regions of the genome. A preference for localized transposition may also have influenced the evolution of the antigen receptor loci
— id: 37389, year: 2001, vol: 21, page: 4302, stat: Journal Article,

Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase
Purugganan MM; Shah S; Kearney JF; Roth DB
2001 Apr 1;29(7):1638-1646, Nucleic acids research
V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80
— id: 37391, year: 2001, vol: 29, page: 1638, stat: Journal Article,

Separation-of-function mutants reveal critical roles for RAG2 in both the cleavage and joining steps of V(D)J recombination
Qiu JX; Kale SB; Yarnell Schultz H; Roth DB
2001 Jan;7(1):77-87, Molecular cell
The only established physiological function of the V(D)J recombinase, comprising RAG1 and RAG2, is to perform DNA cleavage. The molecular roles of RAG2 in cleavage, the mechanisms used to join the broken DNA ends, and the identity of nuclease(s) that open the hairpin coding ends have been unknown. Site-directed mutagenesis targeting each conserved basic amino acid in RAG2 revealed several separation-of-function mutants that address these questions. Analysis of these mutants reveals that RAG2 helps recognize or cleave distorted DNA intermediates and plays an essential role in the joining step of V(D)J recombination. Moreover, the discovery that some mutants block RAG-mediated hairpin opening in vitro provides a critical link between this biochemical activity and coding joint formation in vivo
— id: 37394, year: 2001, vol: 7, page: 77, stat: Journal Article,

Joining-deficient RAG1 mutants block V(D)J recombination in vivo and hairpin opening in vitro
Yarnell Schultz H; Landree MA; Qiu JX; Kale SB; Roth DB
2001 Jan;7(1):65-75, Molecular cell
The RAG proteins cleave at V(D)J recombination signal sequences then form a postcleavage complex with the broken ends. The role of this complex in end processing and joining, if any, is undefined. We have identified two RAG1 mutants proficient for DNA cleavage but severely defective for coding and signal joint formation, providing direct evidence that RAG1 is critical for joining in vivo and strongly suggesting that the postcleavage complex is important in end joining. We have also identified a RAG1 mutant that is severely defective for both hairpin opening in vitro and coding joint formation in vivo. These data suggest that the hairpin opening activity of the RAG proteins plays an important physiological role in V(D)J recombination
— id: 37395, year: 2001, vol: 7, page: 65, stat: Journal Article,

Differential requirements for cis and trans V(D)J cleavage: effects of substrate length
Huye LE; Roth DB
2000 Dec 15;28(24):4903-4911, Nucleic acids research
The assembly of productive synaptic complexes is a critical, but poorly understood, regulatory step in V(D)J recombination. Several lines of evidence suggest that there may be important differences between recombination involving sites situated in cis (on the same DNA molecule) and in trans (on separate molecules). Because biochemical experiments using both purified RAG proteins and crude extracts have failed to detect trans cleavage of plasmid substrates it has been thought that there is a substantial bias against trans synapsis. In conflict with these results are more recent studies showing that purified RAG proteins can catalyze trans cleavage of short oligonucleotide substrates. Furthermore, recent experiments have detected efficient trans cleavage of plasmid substrates in vivo. We sought to investigate why these different systems yield such divergent results. We found that, unexpectedly, the ability of both purified RAG proteins and crude extracts to cleave DNA substrates in trans is a function of substrate length. Our data raise two critical issues: first, oligonucleotides, which are the most commonly used substrates to study V(D)J recombination in vitro, do not mimic the behavior of plasmid substrates; second, in the trans cleavage reaction current purified RAG systems do not accurately reflect the in vivo situation. We propose a unifying model to explain the effects of substrate length and coniguration (cis or trans) on the efficiency of synapsis
— id: 37400, year: 2000, vol: 28, page: 4903, stat: Journal Article,

From lymphocytes to sharks: V(D)J recombinase moves to the germline
Roth DB
2000 ;1(2):REVIEWS1014-REVIEWS1014, Genome biology
The antigen-receptor genes of vertebrates are rearranged by a specialized somatic recombination mechanism in developing lymphocytes - and, unexpectedly, also in the germline of cartilaginous fishes. The recombination system that carries out these DNA rearrangements may thus be a significant evolutionary force, perhaps not limited to rearrangements at antigen-receptor loci
— id: 37393, year: 2000, vol: 1, page: REVIEWS1014, stat: Journal Article,

New guardians of the genome
Roth DB; Gellert M
2000 Apr 20;404(6780):823-825, Nature
— id: 37398, year: 2000, vol: 404, page: 823, stat: Journal Article,

Unequal access: regulating V(D)J recombination through chromatin remodeling
Roth DB; Roth SY
2000 Nov 22;103(5):699-702, Cell
— id: 37397, year: 2000, vol: 103, page: 699, stat: Journal Article,

Intermolecular V(D)J recombination is prohibited specifically at the joining step
Han JO; Steen SB; Roth DB
1999 Mar;3(3):331-338, Molecular cell
V(D)J recombination, normally an intramolecular process, assembles immunoglobulin and T cell receptor genes from V, D, and J coding segments. Oncogenic chromosome translocations can result from aberrant rearrangements, such as occur in intermolecular V(D)J recombination. How this is normally prevented remains unclear; DNA cleavage, joining, or both could be impaired when the recombination signal sequences (RSS) are located in trans, on separate DNA molecules. Here, we show that both trans cleavage and joining of signal ends occur efficiently in vivo. Unexpectedly, trans joining of coding ends is severely impaired (100-to 1000-fold), indicating that protection against intermolecular V(D)J recombination is established at the joining step. These findings suggest a novel surveillance mechanism for eliminating cells containing aberrant V(D)J rearrangements
— id: 37405, year: 1999, vol: 3, page: 331, stat: Journal Article,

What prevents intermolecular V(D)J recombination?
Huye LE; Han JO; Roth DB
1999 ;64(7):191-195, Cold Spring Harbor symposia on quantitative biology
— id: 37392, year: 1999, vol: 64, page: 191, stat: Journal Article,

Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination
Landree MA; Wibbenmeyer JA; Roth DB
1999 Dec 1;13(23):3059-3069, Genes & development
RAG1 and RAG2 initiate V(D)J recombination, the process of rearranging the antigen-binding domain of immunoglobulins and T-cell receptors, by introducing site-specific double-strand breaks (DSB) in chromosomal DNA during lymphocyte development. These breaks are generated in two steps, nicking of one strand (hydrolysis), followed by hairpin formation (transesterification). The nature and location of the RAG active site(s) have remained unknown. Because acidic amino acids have a critical role in catalyzing DNA cleavage by nucleases and recombinases that require divalent metal ions as cofactors, we hypothesized that acidic active site residues are likewise essential for RAG-mediated DNA cleavage. We altered each conserved acidic amino acid in RAG1 and RAG2 by site-directed mutagenesis, and examined >100 mutants using a combination of in vivo and in vitro analyses. No conserved acidic amino acids in RAG2 were critical for catalysis; three RAG1 mutants retained normal DNA binding, but were catalytically inactive for both nicking and hairpin formation. These data argue that one active site in RAG1 performs both steps of the cleavage reaction. Amino acid substitution experiments that changed the metal ion specificity suggest that at least one of these three residues contacts the metal ion(s) directly. These data suggest that RAG-mediated DNA cleavage involves coordination of divalent metal ion(s) by RAG1
— id: 37401, year: 1999, vol: 13, page: 3059, stat: Journal Article,

V(D)J recombination catalyzed by mutant RAG proteins lacking consensus DNA-PK phosphorylation sites
Lin JM; Landree MA; Roth DB
1999 Dec;36(18):1263-1269, Molecular immunology
The process of antigen receptor gene rearrangement, V(D)J recombination, involves DNA cleavage by the RAG-1 and RAG-2 proteins. Cleavage generates covalently sealed (hairpin) DNA ends, termed coding ends, which must be opened by an endonuclease prior to joining. Resolution of these hairpin ends requires the activity of the DNA-dependent protein kinase (DNA-PK), a protein kinase whose specific role is yet undetermined. It has been suggested that phosphorylation of one or both RAG proteins by DNA-PK is required to activate or recruit the hairpin-opening nuclease. Furthermore, very recent work has shown that RAG proteins themselves can open hairpins. These data raise the possibility that DNA-PK-mediated phosphorylation of the RAG proteins could regulate the hairpin opening reaction. To test this hypothesis, we constructed mutant versions of RAG-1 and RAG-2 in which all four DNA-PK consensus phosphorylation sites were removed by site-directed mutagenesis. Our data provide conclusive evidence that phosphorylation of these conserved serine/threonine residues is not required for hairpin opening or joining of V(D)J recombination intermediates
— id: 37399, year: 1999, vol: 36, page: 1263, stat: Journal Article,

Modulation of terminal deoxynucleotidyltransferase activity by the DNA-dependent protein kinase
Mickelsen S; Snyder C; Trujillo K; Bogue M; Roth DB; Meek K
1999 Jul 15;163(2):834-843, Journal of immunology
Rare Ig and TCR coding joints can be isolated from mice that have a targeted deletion in the gene encoding the 86-kDa subunit of the Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). However in the coding joints isolated from Ku86-/- animals, there is an extreme paucity of N regions (the random nucleotides added during V(D)J recombination by the enzyme TdT). This finding is consistent with a decreased frequency of coding joints containing N regions isolated from C.B-17 SCID mice that express a truncated form of the catalytic subunit of the DNA-PK (DNA-PKCS). This finding suggests an unexpected role for DNA-PK in addition of N nucleotides to coding ends during V(D)J recombination. In this report, we establish that TdT forms a stable complex with DNA-PK. Furthermore, we show that DNA-PK modulates TdT activity in vitro by limiting both the length and composition of nucleotide additions
— id: 37404, year: 1999, vol: 163, page: 834, stat: Journal Article,

Roles of the "dispensable" portions of RAG-1 and RAG-2 in V(D)J recombination
Steen SB; Han JO; Mundy C; Oettinger MA; Roth DB
1999 Apr;19(4):3010-3017, Molecular & cellular biology
V(D)J recombination is initiated by introduction of site-specific double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catalyze V(D)J recombination, although less efficiently than their full-length counterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and products. We found that while truncated RAG proteins generate lower levels of recombination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates, termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the 'dispensable' regions of the RAG proteins impairs proper processing of recombination intermediates. Furthermore, we found that removal of portions of the dispensable regions of RAG-1 and RAG-2 affects the efficiency of product formation without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in V(D)J recombination
— id: 37406, year: 1999, vol: 19, page: 3010, stat: Journal Article,

Signal joint formation is inhibited in murine scid preB cells and fibroblasts in substrates with homopolymeric coding ends
Sun, T; Ezekiel, U R; Erskine, L; Agulo, R; Bozek, G; Roth, D; Storb, U
1999 Jun;36(8):551-558, Molecular immunology
During B and T lymphocyte development, immunoglobulin and T cell receptor genes are assembled from the germline V, (D) and J gene segments (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). These DNA rearrangements, responsible for immune system diversity, are mediated by a site specific recombination machinery via recognition signal sequences (RSSs) composed of conserved heptamers and nonamers separated by spacers of 12 or 23 nucleotides (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). Recombination occurs only between a RSS with a 12mer spacer and a RSS with a 23mer spacer (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). RAG1 and RAG2 proteins cleave precisely at the RSS-coding sequence border leading to flush signal ends and coding ends with a hairpin structure (Eastman, M., Leu, T., Schatz, D., 1996. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85-88; Roth, D.B., Menetski, J.P., Nakajima, P.B., Bosma, M.J., Gellert, M., 1992. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell 983-991: Roth, D.B., Zhu, C., Gellert. M., 1993. Characterization of broken DNA molecules associated with V(D)J recombination. Proc. Natl. Acad. Sci. USA 90, 10,788-10,792; van Gent, D., McBlane, J.. Sadofsky, M., Hesse, J., Gellert, M., 1995. Initiation of V(D)J recombination in a cell-free system. Cell 81, 925-934). Signal ends join, forming a signal joint. The hairpin coding ends are opened by a yet unknown endonuclease, and are further processed to form the coding joint (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Ad. Immunol. 56, 27-150.) The murine scid mutation has been shown to affect coding joints, but much less signal joint formation. In this study we demonstrate that the murine scid mutation inhibits correct signal joint formation when both coding ends contain homopolymeric sequences. We suggest that this finding may be due to the function of the SCID protein as an assembly component in V(D)J recombination
— id: 111486, year: 1999, vol: 36, page: 551, stat: Journal Article,

Irradiation-mediated rescue of T cell-specific V(D)J recombination and thymocyte differentiation in severe combined immunodeficient mice by bone marrow cells
Wang C; Bogue MA; Levitt JM; Roth DB
1999 Nov 1;190(9):1257-1262, Journal of experimental medicine
In SCID (severe combined immunodeficient) mice, proper assembly of immunoglobulin and T cell receptor (TCR) genes is blocked by defective V(D)J recombination so that B and T lymphocyte differentiation is arrested at an early precursor stage. Treating the mice with gamma irradiation rescues V(D)J rearrangement at multiple TCR loci, promotes limited thymocyte differentiation, and induces thymic lymphomas. These effects are not observed in the B cell lineage. Current models postulate that irradiation affects intrathymic T cell precursors. Surprisingly, we found that transfer of irradiated SCID bone marrow cells to unirradiated host animals rescues both TCR rearrangements and thymocyte differentiation. These data indicate that irradiation affects precursor cells at an earlier stage of differentiation than was previously thought and suggest new models for the mechanism of irradiation rescue
— id: 37403, year: 1999, vol: 190, page: 1257, stat: Journal Article,

Irradiation-induced rescue of thymocyte differentiation and V(D)J recombination in mice lacking the catalytic subunit of DNA-dependent protein kinase
Wang C; Bogue MA; Nguyen AP; Roth DB
1999 Dec 1;163(11):6065-6071, Journal of immunology
Scid mice express a truncated form of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and are unable to properly rearrange their Ig and TCR genes, resulting in a severe combined immunodeficiency that is characterized by arrested differentiation of B and T lymphocytes. Treatment of scid mice with low doses of gamma irradiation rescues rearrangements at several TCR loci and promotes limited thymocyte differentiation. The machinery responsible for sensing DNA damage and the mechanism by which irradiation compensates for the scid defect in TCR recombination remain unknown. Because DNA-PKcs is present in scid thymocytes, it may mediate some or all of the irradiation effects. To test this hypothesis, we examined the effects of irradiation on DNA-PKcs-deficient (slip) mice. Our data provide the first evidence that DNA-PKcs is not required for limited rescue of thymocyte differentiation or TCR rearrangements
— id: 37402, year: 1999, vol: 163, page: 6065, stat: Journal Article,

Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation
Bogue MA; Jhappan C; Roth DB
1998 Dec 22;95(26):15559-15564, Proceedings of the National Academy of Sciences of the United States of America
Previous studies have suggested that ionizing radiation causes irreparable DNA double-strand breaks in mice and cell lines harboring mutations in any of the three subunits of DNA-dependent protein kinase (DNA-PK) (the catalytic subunit, DNA-PKcs, or one of the DNA-binding subunits, Ku70 or Ku86). In actuality, these mutants vary in their ability to resolve double-strand breaks generated during variable (diversity) joining [V(D)J] recombination. Mutant cell lines and mice with targeted deletions in Ku70 or Ku86 are severely compromised in their ability to form coding and signal joints, the products of V(D)J recombination. It is noteworthy, however, that severe combined immunodeficient (SCID) mice, which bear a nonnull mutation in DNA-PKcs, are substantially less impaired in forming signal joints than coding joints. The current view holds that the defective protein encoded by the murine SCID allele retains enough residual function to support signal joint formation. An alternative hypothesis proposes that DNA-PKcs and Ku perform different roles in V(D)J recombination, with DNA-PKcs required only for coding joint formation. To resolve this issue, we examined V(D)J recombination in DNA-PKcs-deficient (SLIP) mice. We found that the effects of this mutation on coding and signal joint formation are identical to the effects of the SCID mutation. Signal joints are formed at levels 10-fold lower than in wild type, and one-half of these joints are aberrant. These data are incompatible with the notion that signal joint formation in SCID mice results from residual DNA-PKcs function, and suggest a third possibility: that DNA-PKcs normally plays an important but nonessential role in signal joint formation
— id: 37407, year: 1998, vol: 95, page: 15559, stat: Journal Article,

V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells
Han JO; Erskine LA; Purugganan MM; Stamato TD; Roth DB
1998 Aug 15;26(16):3769-3775, Nucleic acids research
V(D)J recombination assembles immunoglobulin (Ig) and T cell receptor (TCR) gene segments during lymphocyte development. Recombination is initiated by the RAG-1 and RAG-2 proteins, which introduce double-stranded DNA breaks (DSB) adjacent to the Ig and TCR gene segments. The broken ends are joined by the DSB repair machinery, which includes the XRCC4 protein. While XRCC4 is essential for both DSB repair and V(D)J recombination, the functions of this protein remain enigmatic. Because the rare V(D)J recombination products isolated from XRCC4-deficient cells generally show evidence of excessive nucleotide loss, it was hypothesized that XRCC4 may function to protect broken DNA ends. Here we report the first examination of V(D)J recombination intermediates in XRCC4-deficient cells. We found that both types of intermediates, signal ends and coding ends, are abundant in the absence of XRCC4. Furthermore, the signal ends are full length. We also showed that alternative V(D)J recombination products, hybrid joints, form with normal efficiency and without excessive deletion in XRCC4-deficient cells. These data indicate that impaired formation of V(D)J recombination products in XRCC4-deficient cells does not result from excessive degradation of recombination intermediates. Potential roles of XRCC4 in the joining reaction are discussed
— id: 37410, year: 1998, vol: 26, page: 3769, stat: Journal Article,

Double-strand break repair in Ku86- and XRCC4-deficient cells
Kabotyanski EB; Gomelsky L; Han JO; Stamato TD; Roth DB
1998 Dec 1;26(23):5333-5342, Nucleic acids research
The Ku86 and XRCC4 proteins perform critical but poorly understood functions in the repair of DNA double-strand breaks. Both Ku 86- and XRCC4-deficient cells exhibit profound radiosensitivity and severe defects in V(D)J recombination, including excessive deletions at recombinant junctions. Previous workers have suggested that these phenomena may reflect defects in joining of the broken DNA ends or in protection of the ends from nucleases. However, end joining in XRCC4-deficient cells has not been examined. Here we show that joining of both matched and mismatched DNA ends occurs efficiently in XRCC4-deficient cells. Furthermore, analysis of junctions shows that XRCC4 is not required to protect the ends from degradation. However, nucleotide sequence analysis of junctions derived from joining of mismatched DNA ends in XRCC4-deficient cells revealed a strong preference for a junction containing a 7 nt homology. Similar results were obtained in Ku86-deficient cells. These data suggest that in the absence of XRCC4 or Ku86, joining is assisted by base pairing interactions, supporting the hypothesis that these proteins may participate in aligning or stabilizing intermediates in end joining
— id: 37408, year: 1998, vol: 26, page: 5333, stat: Journal Article,

VDJ recombination: a transposase goes to work
Roth DB; Craig NL
1998 Aug 21;94(4):411-414, Cell
— id: 37409, year: 1998, vol: 94, page: 411, stat: Journal Article,

V(D)J recombination in Ku86-deficient mice: distinct effects on coding, signal, and hybrid joint formation
Bogue MA; Wang C; Zhu C; Roth DB
1997 Jul;7(1):37-47, Immunity
Ku, a heterodimer of 70 and 86 kDa subunits, plays a critical but poorly understood role in V(D)J recombination. Although Ku86-deficient mice are defective in coding and signal joint formation, rare recombination products have been detected by PCR. Here, we report nucleotide sequences of 99 junctions from Ku86-deficient mice. Over 90% of the coding joints, but not signal or hybrid joints, exhibit short sequence homologies, indicating that homology is required to join coding ends in the absence of Ku86. Our results suggest that Ku86 may normally have distinct functions in the formation of these different types of junctions. Furthermore, Ku86(-/-) joints are unexpectedly devoid of N-region diversity, suggesting a novel role for Ku in the addition of N nucleotides by terminal deoxynucleotidyl transferase
— id: 37413, year: 1997, vol: 7, page: 37, stat: Journal Article,

Growth retardation and leaky SCID phenotype of Ku70-deficient mice
Gu Y; Seidl KJ; Rathbun GA; Zhu C; Manis JP; van der Stoep N; Davidson L; Cheng HL; Sekiguchi JM; Frank K; Stanhope-Baker P; Schlissel MS; Roth DB; Alt FW
1997 Nov;7(5):653-665, Immunity
Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components
— id: 37411, year: 1997, vol: 7, page: 653, stat: Journal Article,

Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products
Han JO; Steen SB; Roth DB
1997 Apr;17(4):2226-2234, Molecular & cellular biology
Ku, a heterodimer of 70- and 86-kDa subunits, serves as the DNA binding component of the DNA-dependent protein kinase (DNA-PK). Cells deficient for the 86-kDa subunit of Ku (Ku86-deficient cells) lack Ku DNA end-binding activity and are severely defective for formation of the standard V(D)J recombination products, i.e., signal and coding joints. It has been widely hypothesized that Ku is required for protection of broken DNA ends generated during V(D)J recombination. Here we report the first analysis of V(D)J recombination intermediates in a Ku-deficient cell line. We find that full-length, ligatable signal ends are abundant in these cells. These data show that Ku86 is not required for the protection or stabilization of signal ends, suggesting that other proteins may perform this function. The presence of high levels of signal ends in Ku-deficient cells prompted us to investigate whether these ends could participate in joining reactions. We show that nonstandard V(D)J recombination products (hybrid joints), which involve joining a signal end to a coding end, form with similar efficiencies in Ku-deficient and wild-type fibroblasts. These data support the surprising conclusion that Ku is not required for some types of V(D)J joining events. We propose a novel RAG-mediated joining mechanism, analogous to disintegration reactions performed by retroviral integrases, to explain how formation of hybrid joints can bypass the requirement for Ku and DNA-PK
— id: 37415, year: 1997, vol: 17, page: 2226, stat: Journal Article,

Initiation of V(D)J recombination in vivo: role of recombination signal sequences in formation of single and paired double-strand breaks
Steen SB; Gomelsky L; Speidel SL; Roth DB
1997 May 15;16(10):2656-2664, EMBO journal
In V(D)J recombination, double-strand breaks (DSBs) are introduced at recombination signal sequences (RSSs) which consist of three distinct elements: a heptamer, a 12 or 23 nucleotide spacer and a nonamer. Efficient DSB formation requires a 12/23 RSS pair and occurs at both RSS in a temporally coupled fashion (coupled cleavage). It remains unknown which RSS elements are important for coupled cleavage. Furthermore, it has not been established whether some RSS components are critical only for cleavage in cis, with others mainly promoting cleavage in trans at the partner RSS. We investigated these questions by analyzing the effects of RSS mutations on the formation of DSBs in vivo. The abundance of DSBs in cis (at the mutant RSS) and in trans (at the consensus RSS) was determined using an established ligation-mediated PCR assay. We also developed a Southern blotting approach that allows the first direct measurement of dual and single RSS cleavage in vivo. Our results demonstrate that the heptamer, spacer and nonamer elements are all required for coupled cleavage in vivo. These studies also provide evidence for cleavage events involving a single RSS both in mutant substrates and in substrates containing a consensus 12/23 RSS pair
— id: 37414, year: 1997, vol: 16, page: 2656, stat: Journal Article,

Normal junctional diversification of immune receptors in p53-deficient mice
Wang C; Bogue MA; Roth DB; Meek K
1997 Jul 15;159(2):757-762, Journal of immunology
One of the strategies that the immune system utilizes to generate Ab and TCR diversity is programmed imprecision of coding joint formation. This is accomplished by both nucleotide loss and random nucleotide addition (N segments) to the termini of immune receptor coding segments before resolution. Although it has been known for more than a decade that terminal deoxynucleotidyl transferase is the enzyme responsible for N segment addition, the enzymes responsible for nucleotide loss have not been identified. Recently, the p53 tumor suppressor protein was shown to have an intrinsic exonuclease activity; we reasoned that p53 as an exonuclease might be responsible for coding end processing during V(D)J recombination. Thus, we examined nucleotide loss from Ig and TCR-beta coding joints in mice lacking p53. We find no significant difference in the degree of nucleotide loss in coding joints isolated from these animals as compared with littermate controls. Thus, we conclude that p53 does not play a role in removal of nucleotides from coding termini during V(D)J recombination. Additionally, recent evidence has surfaced suggesting that p53 may play an important checkpoint role in early thymocyte differentiation. More specifically, it has been suggested that p53 is required to prevent thymocytes from maturing to the double-positive stage in the absence of a functionally rearranged TCR-beta allele. Our data suggest that TCR-beta selection is not affected in p53-deficient, V(D)J rearrangement-proficient mice
— id: 37412, year: 1997, vol: 159, page: 757, stat: Journal Article,

Mechanism of V(D)J recombination
Bogue M; Roth DB
1996 Apr;8(2):175-180, Current opinion in immunology
V(D)J recombination can be separated into two basic operations: DNA cleavage and joining of broken ends. Our understanding of both reactions has increased substantially in the past year. Major advances include the development of a cell-free system capable of cleavage and the identification of several proteins involved in both V(D)J recombination and double-strand break repair
— id: 37419, year: 1996, vol: 8, page: 175, stat: Journal Article,

p53 is required for both radiation-induced differentiation and rescue of V(D)J rearrangement in scid mouse thymocytes
Bogue MA; Zhu C; Aguilar-Cordova E; Donehower LA; Roth DB
1996 Mar 1;10(5):553-565, Genes & development
The murine scid mutation affects both V(D)J recombination and DNA repair. This mutation has been mapped to the gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PK), which is activated by DNA damage in normal cells. In scid mice, antigen receptor gene rearrangements are initiated normally, but impaired joining of coding ends prevents assembly of functional receptor genes, resulting in arrest of B- and T-cell development. Others have shown that exposure of scid mice to genotoxic agents such as gamma-irradiation rescues rearrangement at the T-cell receptor (TCR) beta locus and promotes thymocyte development. Here we demonstrate that irradiation rescues rearrangements at multiple TCR loci, suggesting a general effect on the recombination mechanism. Furthermore, our data show that p53 is required for irradiation-mediated rescue of both thymocyte development and V(D)J recombination. We also find that thymocyte proliferation and differentiation in the absence of DNA damage do not require p53 and are not sufficient to rescue V(D)J recombination. These results suggest that exposure to ionizing radiation facilitates a partial bypass of the scid defect, perhaps by inducing p53-dependent DNA damage response pathways
— id: 37420, year: 1996, vol: 10, page: 553, stat: Journal Article,

High-frequency illegitimate integration of transfected DNA at preintegrated target sites in a mammalian genome
Merrihew RV; Marburger K; Pennington SL; Roth DB; Wilson JH
1996 Jan;16(1):10-18, Molecular & cellular biology
To examine the mechanisms of recombination governing the illegitimate integration of transfected DNA into a mammalian genome, we developed a cell system that selects for integration events in defined genomic regions. Cell lines with chromosomal copies of the 3' portion of the adenine phosphoribosyltransferase (APRT) gene (targets) were established. The 5' portion of the APRT gene, which has no homology to the integrated 3' portion, was then electroporated into the target cell lines, and selection for APRT gene function was applied. The reconstruction of the APRT gene was detected at frequencies ranging from less than 10(-7) to 10(-6) per electroporated cell. Twenty-seven junction sequences between the integrated 5' APRT and its chromosomal target were analyzed. They were found to be randomly distributed in a 2-kb region immediately in front of the 3' portion of the APRT gene. The junctions fell into two main classes: those with short homologies (microhomologies) and those with inserted DNA of uncertain origin. Three long inserts were shown to preexist elsewhere in the genome. Reconstructed cell lines were analyzed for rearrangements at the target site by Southern blotting; a variety of simple and complex rearrangements were detected. Similar analysis of individual clones of the parental cell lines revealed analogous types of rearrangement, indicating that the target sites are unstable. Given the high frequency of integration events at these sites, we speculate that transfected DNA may preferentially integrate at unstable mammalian loci. The results are discussed in relation to possible mechanisms of DNA integration
— id: 37423, year: 1996, vol: 16, page: 10, stat: Journal Article,

The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo
Steen SB; Gomelsky L; Roth DB
1996 Jun;1(6):543-553, Genes to cells
BACKGROUND: V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this '12/23 rule' regulates cleavage, joining, or both. RESULTS: We describe a novel system that permits semiquantitative detection of DSB at RSS derived from V(D)J recombination substrates transfected into cultured cells. This approach provides a powerful new tool for analysis of the cleavage and joining steps of V(D)J recombination in vivo. In this study, substrates containing either a consensus 12/23 RSS pair or various deviations from the consensus were used to investigate the requirements for cleavage. The results show that both a 12-spacer and a 23-spacer RSS are required for efficient cleavage. Truncated RAG-1 and RAG-2 proteins, while capable of cleaving at isolated RSS in cell-free systems, also require a 12/23 RSS pair for efficient cleavage in vivo. CONCLUSIONS: These results suggest that the 12/23 rule is enforced at or prior to cleavage and support a synapsis model for V(D)J recombination. Detection of rare cleavage events in substrates containing a single RSS or a pair of RSS with the same spacer length provide evidence for an inefficient, single RSS cleavage pathway that may contribute to aberrant V(D)J rearrangements in vivo
— id: 37418, year: 1996, vol: 1, page: 543, stat: Journal Article,

Double-strand breaks, DNA hairpins, and the mechanism of V(D)J recombination
Steen SB; Zhu C; Roth DB
1996 ;217(5):61-77, Current topics in microbiology & immunology
— id: 37422, year: 1996, vol: 217, page: 61, stat: Journal Article,

Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates
Zhu C; Bogue MA; Lim DS; Hasty P; Roth DB
1996 Aug 9;86(3):379-389, Cell
Ku is a heterodimeric DNA end binding complex composed of 70 and 86 kDa subunits. Here, we show that Ku86 is essential for normal V(D)J recombination in vivo, as Ku86-deficient mice are severely defective for formation of coding joints. Unlike severe combined immunodeficient (scid) mice, Ku86-deficient mice are also defective for signal joint formation. Both hairpin coding ends and blunt full-length signal ends accumulate. Contrary to expectation, Ku86 is evidently not required for protection of either type of V(D)J recombination intermediate. Instead, V(D)J recombination appears to be arrested after the cleavage step in Ku86-deficient mice. We suggest that Ku86 may be required to remodel or disassemble DNA-protein complexes containing broken ends, making them available for further processing and joining
— id: 37417, year: 1996, vol: 86, page: 379, stat: Journal Article,

Thymocyte differentiation in gamma-irradiated severe-combined immunodeficient mice: characterization of intermediates and products of V(D)J recombination at the T cell receptor alpha locus
Zhu C; Bogue MA; Roth DB
1996 Dec;26(12):2859-2865, European journal of immunology
Treatment with DNA-damaging agents promotes rescue of V(D)J recombination, limited thymocyte differentiation, and development of thymic lymphomas in severe-combined immunodeficient (SCID) mice. One intriguing aspect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) beta, gamma and delta loci, but not at the TCR alpha locus. Current models posit that only those loci that are recombinationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation-mediated polymerase chain reaction assays to detect a specific class of recombination intermediates, hairpin coding ends, at the TCR alpha locus. We found that J alpha-coding ends are undetectable in unirradiated SCID thymocytes, but accumulate after irradiation at times coincident with the emergence of a CD4+ CD8+ thymocyte population. Coding joints produced by joining of these ends, however, are extremely rare. To test whether the presence of hairpin coding ends at TCR alpha is sufficient for irradiation-mediated rescue of coding joint formation, we administered a second dose of gamma-irradiation after abundant CD4+ CD8+ thymocytes and hairpin TCR alpha coding ends had accumulated. This treatment failed to stimulate rescue of TCR alpha coding joints. Thus, the presence of hairpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results support a refined model for irradiation-mediated rescue of TCR rearrangements in SCID mice
— id: 37416, year: 1996, vol: 26, page: 2859, stat: Journal Article,

Mechanism of V(D)J recombination
Zhu C; Roth DB
1996 ;28(5):295-309, Cancer surveys
THe V(D)J recombination reaction can be separated into two fundamental operations: site specific cleavage and joining of broken ends. Much has been learned about the mechanisms of these steps in the past few years. Recent experiments have shown that cleavage is catalyzed by the RAG1 and RAG2 proteins and generates an asymmetric set of broken ends: hairpin coding ends and blunt signal ends. A cell free system capable of performing cleavages has been established, and detailed biochemical information about the reaction should accumulate rapidly. In vivo studies have provided insights into the regulation of cleavage. Recent experiments using artificial recombination substrates in cultured cells have shown that efficient cleavage requires a pair of RSS, one with a 12 nucleotide spacer and one with a 23 nucleotide spacer. Our understanding of the joining mechanism has also increased substantially, as several proteins involved in coding joint and signal joint formation (as well as in DSB repair) were recently identified
— id: 37421, year: 1996, vol: 28, page: 295, stat: Journal Article,

Hairpin opening by single-strand-specific nucleases
Kabotyanski EB; Zhu C; Kallick DA; Roth DB
1995 Oct 11;23(19):3872-3881, Nucleic acids research
DNA molecules with covalently sealed (hairpin) ends are probable intermediates in V(D)J recombination. According to current models hairpin ends are opened to produce short single-stranded extensions that are thought to be precursors of a particular type of extra nucleotides, termed P nucleotides, which are frequently present at recombination junctions. Nothing is known about the activities responsible for hairpin opening. We have used two single-strand-specific nucleases to explore the effects of loop sequence on the hairpin opening reaction. Here we show that a variety of hairpin ends are opened by P1 nuclease and mung bean nuclease (MBN) to leave short, 1-2 nt single-stranded extensions. Analysis of 22 different hairpin sequences demonstrates that the terminal 4 nt of the hairpin loop strongly influence the sites of cleavage. Correlation of the nuclease digestion patterns with structural (NMR) data for some of the hairpin loops studied here provides new insights into the structural features recognized by these enzymes
— id: 37425, year: 1995, vol: 23, page: 3872, stat: Journal Article,

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage
Nakajima PB; Menetski JP; Roth DB; Gellert M; Bosma MJ
1995 Nov;3(5):609-621, Immunity
The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement
— id: 37424, year: 1995, vol: 3, page: 609, stat: Journal Article,

Repair and recombination. How to make ends meet
Roth DB; Lindahl T; Gellert M
1995 May 1;5(5):496-499, Current biology. CB
The repair of double-stranded breaks in DNA and the recombination of antibody gene V(D)J segments share a common pathway involving the Ku protein, which binds DNA ends, and its associated protein kinase
— id: 37426, year: 1995, vol: 5, page: 496, stat: Journal Article,

Characterization of coding ends in thymocytes of scid mice: implications for the mechanism of V(D)J recombination
Zhu C; Roth DB
1995 Jan;2(1):101-112, Immunity
We previously identified possible intermediates in V(D)J recombination at the TCR delta locus and characterized molecules with signal ends and with covalently sealed (hairpin) coding ends in thymocytes of scid mice by Southern blotting. Here, we use a sensitive ligation-mediated PCR assay to demonstrate that all coding ends detected in scid thymocytes are covalently sealed. Neither coding nor signal ends exhibit loss or addition of nucleotides. These data imply that hairpin formation is coupled to the initial cleavage at the signal/coding border, and that the cleavage step in V(D)J recombination is conservative. In scid/+ or wild-type thymocytes, hairpin coding ends are at least 1000-fold less abundant than signal ends. These results provide insight into the mechanism of V(D)J recombination
— id: 37427, year: 1995, vol: 2, page: 101, stat: Journal Article,

Characterization of broken DNA molecules associated with V(D)J recombination
Roth DB; Zhu C; Gellert M
1993 Nov 15;90(22):10788-10792, Proceedings of the National Academy of Sciences of the United States of America
We previously demonstrated that DNA molecules with double-strand breaks at variable-(diversity)-joining [V(D)J] recombination signal sequences are relatively abundant in mouse thymocytes. This abundance strongly suggests that the mechanism of V(D)J recombination involves double-strand cleavage at recombination signals. As a first step toward understanding the mechanism of cleavage, we used a sensitive PCR assay to characterize the structure of one class of cleavage products, the signal ends, in detail. Here we demonstrate that most of these ends are blunt and terminate in 5' phosphoryl groups. Virtually all of the flush signal ends are full length. A minor subpopulation of broken ends terminates in short single-strand extensions. We have found no evidence for covalent DNA-protein linkages involving the signal ends. These data allow further refinement of the double-strand cleavage model for V(D)J recombination
— id: 37428, year: 1993, vol: 90, page: 10788, stat: Journal Article,

V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes
Roth DB; Menetski JP; Nakajima PB; Bosma MJ; Gellert M
1992 Sep 18;70(6):983-991, Cell
Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor
— id: 37429, year: 1992, vol: 70, page: 983, stat: Journal Article,

Double-strand breaks associated with V(D)J recombination at the TCR delta locus in murine thymocytes
Roth DB; Nakajima PB; Menetski JP; Bosma MJ; Gellert M
1992 ;182(1):115-124, Current topics in microbiology & immunology
— id: 37431, year: 1992, vol: 182, page: 115, stat: Journal Article,

V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals
Roth DB; Nakajima PB; Menetski JP; Bosma MJ; Gellert M
1992 Apr 3;69(1):41-53, Cell
In the murine T cell receptor delta locus, V(D)J recombination events frequently involve the D2 and J1 elements. Here we report the presence of double-strand breaks at recombination signals flanking D2 in approximately 2% of thymus DNA. An excised linear species containing the sequences between D2 and J1 and a circular product of the joining of D2 and J1 recombination signals were also found. Although broken molecules with signal ends were detected, no species with coding ends could be identified. Observation of these broken molecules in thymus, but not in liver or spleen, provides the first direct evidence for an association between specific cleavage of chromosomal DNA and recombination in mammalian cells, and supports a breakage-reunion model of V(D)J recombination
— id: 37430, year: 1992, vol: 69, page: 41, stat: Journal Article,

Oligonucleotide capture during end joining in mammalian cells
Roth DB; Proctor GN; Stewart LK; Wilson JH
1991 Dec;19(25):7201-7205, Nucleic acids research
Extra nucleotides (termed filler DNA) are found at about 10% of the junctions of the genetic rearrangements that arise by illegitimate recombination in mammalian cells. Such filler DNAs could arise by the joining of oligonucleotide fragments to broken ends prior to end joining. We tested this possibility by microinjecting mixtures of defined oligonucleotides with SV40 genomes that were linearized in the intron for T antigen, a site where incorporation of extra nucleotides does not impair viability. Using an injection ratio of 1000 oligonucleotides per DNA end, we screened viable genomes for incorporation of single-stranded and double-stranded oligonucleotides with varying degrees of complementarity to the ends of the linear SV40 molecules. Genomes from 510 independent plaques were screened by restriction digestion to identify those that had picked up a restriction site unique to the injected oligonucleotides. Double-stranded oligonucleotides that were fully complementary to the SV40 ends were readily incorporated, but uptake of the other oligonucleotides was not detected by restriction analysis. Nucleotide sequences of junctions from 12 genomes derived from co-injection of noncomplementary oligonucleotides revealed two with filler DNA, but neither could be assigned unambiguously to the injected oligonucleotides
— id: 37432, year: 1991, vol: 19, page: 7201, stat: Journal Article,

Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation
Roth DB; Chang XB; Wilson JH
1989 Jul;9(7):3049-3057, Molecular & cellular biology
Extra nucleotides (termed filler DNA) are commonly found at the junctions of genetic rearrangements in mammalian cells. The filler DNA at immune system rearrangements, which are called N regions, are generated at VDJ joints primarily by terminal deoxynucleotidyl transferase. However, the origin of filler DNA at genetic rearrangements in nonlymphoid cells is uncertain. In an analysis of more than 200 junctions that arose by circularization of transfected linear DNA (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986), we found 18 junctions with extra nucleotides exactly at the point of circularization. Analysis of these 18 junctions indicated that nonlymphoid cells could add extra nucleotides to the ends of duplex DNA. The characteristics of the extra nucleotides at these junctions and at 31 other rearrangement junctions from nonlymphoid cells were quite similar, suggesting that many genetic rearrangements may pass through a stage with free DNA ends. A comparison of the filler DNA at these 49 nonimmune system rearrangements with 97 N regions derived from immune system rearrangements suggested that lymphoid and nonlymphoid cells use different mechanisms for insertion of filler DNA, as expected from the absence of detectable terminal deoxynucleotidyl transferase in cells from nonlymphoid tissues. The filler DNAs at a smaller group of 22 translocations associated with cancer had features in common with both immune and nonimmune system rearrangements and therefore may represent a mixture of these two processes. Mechanisms that might account for the presence of filler DNA in nonlymphoid cells are discussed
— id: 37433, year: 1989, vol: 9, page: 3049, stat: Journal Article,

Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction
Roth DB; Wilson JH
1986 Dec;6(12):4295-4304, Molecular & cellular biology
Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed
— id: 37434, year: 1986, vol: 6, page: 4295, stat: Journal Article,

Mechanisms of nonhomologous recombination in mammalian cells
Roth DB; Porter TN; Wilson JH
1985 Oct;5(10):2599-2607, Molecular & cellular biology
The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed
— id: 37435, year: 1985, vol: 5, page: 2599, stat: Journal Article,

Relative rates of homologous and nonhomologous recombination in transfected DNA
Roth DB; Wilson JH
1985 May;82(10):3355-3359, Proceedings of the National Academy of Sciences of the United States of America
Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-base-pair duplication at its termini. Once inside the cell, this molecule must circularize to initiate lytic infection. Circularization can occur either by direct, nonhomologous end-joining or by homologous recombination within the duplicated region. Although the products of the two recombination pathways are different, they are equally infectious. Since homologous and nonhomologous recombination processes are competing for the same substrate, the relative amounts of the products of each pathway should reflect the relative rates of homologous and nonhomologous recombination. Analysis of individual recombinant genomes from 164 plaques indicates that the rate of circularization by nonhomologous recombination is 2- to 3-fold higher than the rate of homologous recombination. The assay system described here may prove to be useful for testing procedures designed to influence the relative rates of homologous and nonhomologous recombination
— id: 37436, year: 1985, vol: 82, page: 3355, stat: Journal Article,

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli
Yeung, C Y; Ingolia, D E; Roth, D B; Shoemaker, C; Al-Ubaidi, M R; Yen, J Y; Ching, C; Bobonis, C; Kaufman, R J; Kellems, R E
1985 Aug 25;260(18):10299-10307, Journal of biological chemistry
Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%
— id: 111485, year: 1985, vol: 260, page: 10299, stat: Journal Article,