Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking

Eastman, Quinn; Villey, Isabelle; Schatz, David G.

doi:10.1128/mcb.19.5.3788

Cited by 72 publications

(72 citation statements)

References 57 publications

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“…A RAG complex minimally containing a RAG-1 dimer and monomeric RAG-2 associates with one RSS (a 12-RSS is shown here for simplicity) and then captures an appropriate RSS partner (31). RAG-1 interacts with both the nonamer (long hatched box) and the heptamer (short hatched box), and RAG-2 is localized at the heptamer-coding junction (12,48,49). The second RAG-2 subunit may associate with the complex either before or after acquisition of the second RSS (in this case, a 23-RSS).…”

Section: Vol 22 2002 Assembly and Activity Of Rag Synaptic Complexementioning

confidence: 99%

A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals

Swanson

2002

Molecular and Cellular Biology

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Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.The antigen binding regions of immunogobulin and T-cell receptors are encoded in arrays of noncontiguous coding segments, called variable (V), diversity (D), and joining (J), that are assembled during lymphocyte development to generate the variable-region exon of the mature antigen receptor gene (5). The assembly process, termed V(D)J recombination, involves a series of site-specific DNA rearrangements mediated by recombination signal sequences (RSSs) abutting the coding segments. Each RSS contains conserved heptamer and nonamer elements, separated by nominally conserved spacer DNA that is either 12 or 23 bp long (12-RSS and 23-RSS, respectively). The RSS facilitates the generation of functional antigen receptors via the "12/23 rule," a restriction that limits recombination to a pair of coding segments whose flanking RSSs bear spacer DNAs of different lengths.The products of recombination-activating genes 1 and 2, RAG-1 and RAG-2 (35, 42), initiate V(D)J recombination by introducing a DNA double-strand break (DSB) at a 12/23 pair of RSSs (at the heptamer-coding segment border). The cleavage reaction generates two distinct DNA intermediates: blunt, 5Ј-phosphorylated signal ends (SE) and coding ends terminating in covalently sealed DNA hairpin structures (38,39,43). DSB intermediates are generated in two biochemical steps: first-strand nicking at the 5Ј end of the heptamer, followed by a direct transesterification reaction in which the 3Ј-OH exposed in the nicking step attacks the phosphodiester on the antiparallel DNA strand (26, 52). The RAG proteins also mediate other reactions in vitro, including a disintegratio...

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Section: Vol 22 2002 Assembly and Activity Of Rag Synaptic Complexementioning

confidence: 99%

A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals

Swanson

2002

Molecular and Cellular Biology

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“…The upregulation/lack of downregulation of the respective DNA damage response genes, XPF and UV-RAG after Cr exposure in B-5Cr cells is intriguing, particularly in light of their association with nucleotide excision repair (NER) and recombination pathways [76][77][78][79]. We and others have shown that both NER and homologous recombination repair mechanisms are associated with the repair of Cr-induced DNA lesions [80,81] (for review, see [26]).…”

Section: Discussionmentioning

confidence: 99%

Resistance to apoptosis, increased growth potential, and altered gene expression in cells that survived genotoxic hexavalent chromium [Cr(VI)] exposure

et al. 2005

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Certain hexavalent chromium [Cr(VI)] compounds are known genotoxic respiratory carcinogens, which induce apoptosis as a predominant mode of cell death. Selection of cells that are resistant to apoptosis may be a factor in tumour progression. We developed sub-populations of telomerasetransfected human fibroblasts (BJ-hTERT) that survived a 99% clonogenically lethal exposure to Cr (VI) (B-5Cr). B-5Cr cells were markedly resistant to apoptosis induced by several agents and exhibited increased clonogenic survival, especially at apoptogenic doses. B-5Cr cells did not exhibit altered cellular uptake of Cr(VI) and retained a normal p53 response to Cr(VI) exposure. We conducted large-scale gene expression analysis at different time-points after a secondary genotoxic Cr(VI) insult in B-5Cr and BJ-hTERT cells using Affymetrix Genechip® human genome arrays. Cr (VI) exposure led to differential regulation of many genes, which affect a diverse set of cellular activities such as transcription, signal transduction, stress response, cell adhesion, DNA repair, apoptosis and cell cycle modulation. We compared Cr(VI)-induced altered gene expression in the B-5Cr cells to that in the parental cells and identified 223, 147 and 204 genes with at least a two-fold difference in expression at 4, 8 and 18 h after exposure, respectively. Cluster analysis by gene function revealed altered expression of genes involved in apoptosis, cell cycle regulation and DNA repair. Our data suggest an alteration in gene expression that may favor cell survival and/or incomplete DNA repair after genotoxic exposure. Selection of cells with altered expression of these genes may constitute the early stages of tumour progression.

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“…Such oxidized bases tend to be displaced from the helix. Furthermore, cross-linking experiments using iodopyrimidine-modified bases across the codingheptamer region identified C1b (and S2b) as forming crosslinks to RAG1 (35). Although specific amino acid-base contacts were not identified with these methods, it appears that coding flank interactions (at C2t) are occurring in the C-terminal domain between amino acids 889 and 974 (36).…”

Section: Changes In Base Contacts and Complex Conformation During Cleav-mentioning

confidence: 97%

Requirements for DNA hairpin formation by RAG1/2

Grundy

Hesse²,

Gellert³

2007

Proc. Natl. Acad. Sci. U.S.A.

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The rearrangement of antigen receptor genes is initiated by doublestrand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some ''cut-and-paste'' transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation.immunoglobulin gene ͉ recombination ͉ transposase I n many vertebrates, the vast antigen-binding repertoire of immunoglobulins and T cell receptors is created by combinatorial V(D)J recombination using an array of variable (V), diversity (D), and joining (J) gene segments in B and T cell genomic DNA (1). A complex of the RAG1 and RAG2 gene products is responsible for initiating DNA cleavage and probably for mediating the recruitment of nonhomologous end-joining factors to process and join the broken V, (D), and J segments.The sites of DNA cleavage are directed by recombination signal sequences (RSSs) that flank each coding segment. The two types of RSSs (denoted 12RSS and 23RSS) consist of conserved heptamer and nonamer motifs separated by a spacer of 12 or 23 nonconserved base pairs. Normally, DNA cleavage occurs when the RAG complex recognizes and pairs a 12RSS and a 23RSS, thus ensuring the correct recombination of a V to a J or of a V to a D and of a D to a J segment (termed the ''12/23 rule'') (2). Ordered assembly usually begins with RAG1/2 binding to the 12RSS, followed by capture of free 23RSS (3, 4). Changes in the sensitivity of a 12RSS to chemical modification occur upon RAG1/2 binding, consistent with some unwinding at the heptamer-coding border (5, 6). Double-strand breaks at the coding-RSS border are produced by a nick 5Ј of the heptamer; nucleophilic attack on the opposing strand by the 3Ј hydroxyl group at the nick then creates a hairpin by a transesterification reaction (7). The resulting double-strand break consists of a hairpin on the coding flank and a blunt-ended signal sequence. In the presence of Mg 2ϩ , the complete reaction takes place only in the presence of an RSS pair (coupled cleavage) (8, 9). The mechanism of DNA cleavage has been characterized in vitro, mainly with truncated RAG proteins that retain activity but are mor...

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Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking

Cited by 72 publications

References 57 publications

A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals

A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals

Resistance to apoptosis, increased growth potential, and altered gene expression in cells that survived genotoxic hexavalent chromium [Cr(VI)] exposure

Requirements for DNA hairpin formation by RAG1/2

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