Characterization of a Tn 5 pre-cleavage synaptic complex 1 1Edited by M. Gottesman

Bhasin, Archna; Goryshin, Igor Y.; Steiniger-White, Mindy; York, Dona; Reznikoff, William S.

doi:10.1006/jmbi.2000.4048

Cited by 52 publications

(72 citation statements)

References 36 publications

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“…These include (i) the generation of DNA hairpin intermediates (7), (ii) the presence of a single enzyme binding site at each DNA end undergoing cleavage (36), and (iii) the structural resemblance between RAG-1 and the Tn5 transposase with respect to the spacing and organization of critical active-site carboxylate residues (the DDE motif) (33,46). The similarity between RAG-1 and the Tn5 transposase is shown here to extend to the dimeric configuration of both proteins within synaptic complexes (8,10). However, an important distinction between the two recombination systems is that only one protein, the transposase, is necessary to catalyze all reactions underlying Tn5 transposition, whereas RAG-1 requires RAG-2 for V(D)J recombinase activity.…”

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

confidence: 77%

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: 77%

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 excision and insertion involve four catalytic steps at the transposon ends: transferred strand nicking, hairpin formation, hairpin resolution, and strand transfer into target DNA (2). These reactions occur within a protein-DNA complex, termed a synaptic complex, consisting of two Tnp molecules and two end sequences (3,4). Single Tnp molecules bound to single end sequences are not catalytically competent, thus restricting the transposition reactions to synaptic complexes (4,5).…”

Section: Tn5 Transposase (Tnp)mentioning

confidence: 99%

Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

Peterson

Reznikoff

2003

Journal of Biological Chemistry

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Tn5 transposase (Tnp), a 53.3-kDa protein, enables the movement of transposon Tn5 by a conservative mechanism. Within the context of a protein and DNA synaptic complex, a single Tnp molecule catalyzes four sequential DNA breaking and joining reactions at the end of a single transposon. The three amino acids of the DDE motif (Asp-97, Asp-188, and Glu-326), which are conserved among transposases and retroviral integrases, have been shown previously to be absolutely required for all catalytic steps. To probe the effect of active site geometry on the ability to form synaptic complexes and perform catalysis, single mutations at each position of the DDE motif were constructed. The aspartates were changed to glutamates, and the glutamate was changed to an aspartate. These mutants were studied by performing in vitro binding assays using short oligonucleotide substrates simulating the natural substrates for the synaptic complex formation and subsequent transposition steps. The results indicate that the aspartate to glutamate mutations restrict synaptic complex formation with substrates resembling the natural transposon prior to transferred strand nicking. This suggests a structural model in which the donor backbone DNA, prior to nicking, occupies the same space that is invaded by the longer side chains present in the aspartate to glutamate mutants. Additionally, catalytic assays support the previous proposal that the active site coordinates two divalent metal ions.

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“…First, it is believed that each Tnp monomer binds to two separate recognition sequences, forming the initial contacts. The monomers then dimerize to form the synaptic complex (6). This complex brings the end sequences proximal to one another.…”

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confidence: 99%

Mutational Analysis of the Base Flipping Event Found in Tn5 Transposition

Ason

Reznikoff

2002

Journal of Biological Chemistry

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To study the biochemical significance of this phenomenon, we mutated the Tnp residues proposed to be involved in stabilizing this interaction and removed the T2 nucleotide to examine which steps in the transposition reaction require T2-Tnp interactions. From this work, we have determined that stacking interactions between T2 and Tnp are critical for efficient transposition in vitro. In addition, our results suggested that T2-Tnp interactions facilitate hairpin formation and hairpin resolution primarily through base stacking and that T2 plays a role in the alignment of the transposon DNA for strand transfer.Mobile DNA elements share many similar characteristics. The proteins responsible for movement of these elements manifest common mechanistic features and contain considerable structural similarities in their catalytic domains. In the Tn5 system, Tn5 transposase (Tnp) 1 functions as a homodimer and catalyzes all of the steps involved in the movement of Tn5 from one location to another within a genome (3-5).The model for Tn5 transposition is illustrated in Fig. 1. First, it is believed that each Tnp monomer binds to two separate recognition sequences, forming the initial contacts. The monomers then dimerize to form the synaptic complex (6). This complex brings the end sequences proximal to one another. In the synaptic complex, each Tnp monomer binds to both end sequences. The first set of contacts (cis contacts) are maintained predominately between the N-terminal domain and the middle of one end sequence, whereas the second set of Tnp-DNA contacts (trans contacts) are formed primarily between the catalytic domain and the other end sequence (1). This complex is a prerequisite to cleavage, since residues near the trans Tnp-DNA contacts are responsible for strand cleavage (1, 7). The cleavage reaction begins as Tnp nicks the transferred DNA strand at the donor DNA-transposon junction, adjacent to position 1 of the end sequence, leaving a 3Ј OH group. This nick occurs through the nucleophilic attack by an activated water molecule coordinated by Mg 2ϩ . The 3Ј OH group on the transferred strand in turn acts as a nucleophile to cleave the complementary nontransferred DNA strand, forming a hairpin at the transposon end and releasing the donor DNA. Hydrolysis of the hairpin by another activated water molecule, coordinated in the same Tnp monomer, resolves the hairpin and results in the formation of transposon DNA competent for integration (8). The cleaved transposon DNA remains in a nucleoprotein complex called the post-cleavage synaptic complex. This complex binds to the target DNA, forming the target capture complex (9). The transposon DNA contains free 3Ј OH moieties on the transferred strands that attack the phosphodiester backbone of the target DNA in a staggered transesterification reaction termed strand transfer. A 9-bp stagger for the strand transfer step creates two complementary 9-base single-stranded DNA regions flanking the Tnp insertion. These single-stranded DNA regions are repaired by the cell DNA replicat...

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Characterization of a Tn 5 pre-cleavage synaptic complex 1 1Edited by M. Gottesman

Cited by 52 publications

References 36 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

Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

Mutational Analysis of the Base Flipping Event Found in Tn5 Transposition

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