See Preview - "How Homologous Recombination is Initiated: Unexpected Evidence for Single-Strand nicks from V(D)J Site-Specific Recombination" by G. Smith [click here]

2004

G.S. Lee, M B. Brandt, V.L., and D.B. Roth (2004). B Cell Development Leads Off with a Base Hit: dU:dG Mismatches in Class Switching and Hypermutation. Molecular Cell Vol 16, 1-20 [additional figures 1 and 2]

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 funcion in both SHM and CSR.

G.S. Lee, M B. Neiditch, S.S. Salus, and D.B. Roth (2004). RAG Proteins Shepherd Double-Strand Breaks to a Specific Pathway, Suppressing Error-Prone Repair, but RAG Nicking Initiates Homologous Recombination. Cell Apr 117, 171-184.

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.

J.E. Posey, V.L. Brandt, and D.B. Roth (2004). Paradigm switching in the germinal center. Nature Immunology, News and Views, Vol 5, p477

Antigen receptor genes are assembled by a sequence of lineage-specific recombinatorial events, in which DNA breaks must be properly repaired to ensure cell survival and further developmental maturation. B lymphocytes, it seems, use multiple uniqe pathways to repair their DNA

Z. Sandor, M.L. Calicchio, R.G. Sargent, D.B. Roth, and J.H. Wilson (2004). Distinct requirements for Ku and N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks. Nucleic Acids Research, Vol 32. No. 6, p.1866-1873.

2003

Roth, D.B. (2003). Restraining the V(D)J recombinase. Nature Reviews Immunology 3: 656-666 .

Chromosomal 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 lymphocyte development. This is a daring strategy, for 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, since the possibility of transposition brings in an entirely new set of risks. This review discusses mechanisms that might constrain the dangerous potential of the recombinase and preserve genomic integrity during immune system development.

2002

Neiditch, M.B., G.S. Lee, L.E. Huye, V.L. Brandt, and D.B. Roth.  The V(D)J recombinase efficiently cleaves and transposes signal joints. Molecular Cell 9: 871-878.

Signal joints have been considered inert, dead-end products that safeguard the genome from inappropriate rearrangements due to illegitimate joining of signal ends or transposition. We demonstrated that signal joints are not at all inert: they are readily cleaved in vivo and form an excellent substrate for transposition! Furthermore, the RAG proteins can create a double-strand break at a signal joint by an entirely novel, heretofore unsuspected nick-nick mechanism. Our work not only shatters the reigning dogma in the field but raises a very important biological question: how are these ends prevented from engaging in inappropriate rearrangements?

Huye, L.E., Purugganan, M.M., and D.B. Roth, Mutagenesis of all conserved positive charges in RAG-1 reveals catalytic, step-arrest, and separation of function mutants in the V(D)J recombinase.  Mol. Cell. Biol. 22:3460-3473. (See commentary, ASM News, 68, p.344, July, 2002; “Best of ASM” paper.).

This work identifies a new DNA binding domain in RAG-1 that is important for binding to the coding flank, and also describes the first RAG-1 mutants with defects in coding joint formation that form abnormal junctions.  These coding joints show aberrant deletions, short sequence homologies, and excessive P nucleotide insertions – the same features found in mutant cells lacking the DNA-dependent protein kinase.  These mutants firmly implicate the RAG proteins in the joining of coding ends. 

Lee, G.S., M.B. Neiditch, R.R. Sinden, D.B. Roth. (2002) Targeted transposition by the V(D)J recombinase.  Mol. Cell. Biol., 22: 2068-2077.

The first evidence that transposition can be targeted to certain sequence elements.  We found that transposition is stimulated by, and targeted to, distorted DNA.  We also describe a limited form of transposition that occurs in living cells.  Our data support a new model for chromosome translocations in which transposition joins antigen receptor loci to chromosomal sites bearing certain structural features. 

2001

Landree, M.A., S.B. Kale, and D.B. Roth (2001).  Functional organization of the V(D)J cleavage complex, Mol. Cell. Biol. 21:425-4264.

Catalytically inactive RAG-1 mutants (isolated in our laboratory) were used to define the functional organization of the recombination complex, demonstrating that a single active site can carry out both steps of cleavage, nicking and hairpin formation.

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 suggest that the rate-limiting step for transposition occurs after 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 Ig and TCR 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. 

Qiu, J., Kale, S.B., Schultz, H.Y., and D.B.Roth (2001).  Separation of function mutants reveal critical roles for RAG-2 in both the cleavage and joining steps of V(D)J recombination.  Molecular Cell 7: 77-87.

Schultz, H.Y., M.A. Landree, J. Qiu, S.B. Kale, and D.B. Roth (2001). Joining-deficient RAG-1 mutants block V(D)J recombination in vivo.  Molecular Cell 7: 65-75.

These two papers were the first to identify separation-of-function mutants in the V(D)J recombinase:  mutants that can cleave, but have specific defects in signal joint formation, coding joint formation, or both.  These data provided the first proof that the RAG proteins are involved in the joining steps. Furthermore, some joining-deficient RAG mutants showed defects in both hairpin opening in vitro and coding joint formation in vivo, suggesting that the RAG proteins are indeed involved in opening the hairpin coding ends in vivo.

Kale, S.B., M.A. Landree, and D.B. Roth (2001). Conditional RAG-1 mutants block the hairpin formation step of V(D)J recombination.  Molecular and Cellular Biology 21: 459-466 .

We analyzed two conditional RAG-1 mutants that affect residues quite close in the primary sequence to an active site amino acid (D600), and found that they exhibit severely impaired recombination in the presence of certain cleavage site sequences.  These mutants are specifically defective for 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 generation of a distorted/unwound DNA intermediate that has been implicated in hairpin formation.

1999

Landree, M.A., J.A. Wibbenmeyer, D.B. Roth.  (1999) Mutational analysis of RAG-1 and RAG-2 identifies three active site amino acids in RAG-1 critical for both cleavage steps of V(D)J recombination. Genes and Development 13:3059-3069.

It should be noted that two other groups also reported the presence of two catalytic D residues in RAG-1. The Oettinger laboratory published a paper back-to-back with ours, approaching the problem from a different perspective (analysis of metal binding residues); our two groups shared information prior to publication. The Schatz laboratory also published a limited mutagenesis study the following year that also identified the two D residues. Neither of the other papers examined all the acidic residues in RAG-1 (and thus did not identify the third catalytic residue, E962), nor did they examine RAG-2.

Wang, C.,  M. A. Bogue, J. M. Levitt, and D.B. Roth.  (1999) Cells responsible for irradiation-mediated rescue of T cell-specific V(D)J recombination and thymocyte differentiation in scid mice reside in the bone marrow. J. Exp. Med., 190:1257-1262.

Several years ago Danska et al. published a startling observation in Science: V(D)J recombination and thymocyte development, which are blocked by the murine scid mutation, could be partially restored by treating scid mice with low doses of g-irradiation. Curiously, these phenomena are specific to the T cell lineage; moreover, they are accompanied by the invariable development of thymic lymphomas in the irradiated mice. It had been thought that the cells responsible for the effect reside in the thymus at the time of irradiation, and the prevailing view was that irradiation rescues TCR rearrangements in thymocytes by facilitating a transient bypass of the scid defect, promoting the joining of pre-existing hairpin coding ends.

We employed an adoptive transfer approach to determine whether irradiation of either scid thymocytes or bone marrow cells is sufficient to reproduce the irradiation rescue phenomenon upon transfer of these cells to unirradiated host animals. Our data showed that transfer of irradiated adult scid bone marrow, but not thymocytes, rescues both TCR rearrangements and thymocyte differentiation. Thus, exposure of thymocytes or thymic stromal cells to ionizing radiation is not necessary for the irradiation rescue effect. These results indicate that, surprisingly, the cellular targets of irradiation include early lymphocyte progenitor cells residing in the bone marrow, and led us to propose new models for the mechanism of irradiation rescue. We suggest that irradiation induces persistent changes in early lymphoid precursors that can promote joining of DNA ends created at a later stage in lymphocyte differentiation.

Han, J.-O., S.B. Steen, and D.B. Roth.  (1999) Intermolecular V(D)J recombination is prohibited specifically at the joining step.  Molecular Cell 3:331-338.