Tn5: A Molecular Window on Transposition

Reznikoff, William S.; Bhasin, Archna; Davies, D.R.; Goryshin, Igor Y.; Mahnke, Lisa; Naumann, Todd A.; Rayment, Ivan; Steiniger-White, Mindy; Twining, Sally S.

doi:10.1006/bbrc.1999.1891

Cited by 59 publications

(39 citation statements)

References 45 publications

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“…All four subunits interact specifically with DNA, occupying two of three potential binding sites at each Mu end (29), yet only two of four potential active sites are utilized for the chemical steps involved in Mu transposition (32,54). However, aside from a superficial resemblance in the overall number of protomers in the synaptic complex, V(D)J recombination bears few other similarities to Mu transposition, especially relative to Tn5 (and Tn10) transposition (36), with which it shares more features. 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).…”

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

confidence: 99%

“…However, aside from a superficial resemblance in the overall number of protomers in the synaptic complex, V(D)J recombination bears few other similarities to Mu transposition, especially relative to Tn5 (and Tn10) transposition (36), with which it shares more features. 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).…”

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

confidence: 99%

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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%

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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“…1 is the protein responsible for the mobility of the Tn5 transposon (1). Movement of the transposon occurs via a conservative "cut and paste" mechanism that is mediated by Tnp and 19-bp inverted terminal repeats.…”

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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“…The ability to isolate hyperactive transposase mutants has led to the suggestion that transposases have not evolved for maximal activity (40). Since V(D)J recombination shares mechanistic similarities with transpositional recombination systems that use a cut-and-paste mechanism (19,38), it is likely that the RAG complex is also less active than it potentially could be.…”

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confidence: 99%

Identification and Characterization of a Gain-of-Function RAG-1 Mutant

Kriatchko

Anderson

Swanson

2006

Molecular and Cellular Biology

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RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis. V(D)J recombination is the process by which noncontiguous antigen receptor gene coding segments, called variable (V), diversity (D), and joining (J), are assembled during lymphocyte development to produce the variable region exon of a mature antigen receptor gene (3). V(D)J recombination occurs in two distinct phases. In the first phase, two lymphoid cell-specific proteins called RAG-1 and RAG-2 assemble a multiprotein synaptic complex with two different gene segments through interactions with a conserved recombination signal sequence (RSS) that adjoins each gene segment. Each RSS contains a conserved heptamer and nonamer sequence, separated by either 12 or 23 base pairs of intervening DNA of more varied composition (12-RSS and 23-RSS, respectively). Generally, synaptic complexes are assembled with two RSSs whose spacer lengths are different (the 12/23 rule). Subsequently, the RAG proteins catalyze a DNA double-strand break at each RSS (for reviews, see references 10 and 13), yielding a postcleavage complex containing four DNA ends: two blunt, 5Ј phosphorylated recombination signal ends and two coding ends terminating in DNA hairpin structures (41,42,46). The RAG proteins generate these recombination intermediates by nicking the DNA at the junction between the RSS and the coding sequence and then transferring the resulting 3Ј-OH to the opposing DNA strand by direct transesterification (32,...

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Tn5: A Molecular Window on Transposition

Cited by 59 publications

References 45 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

Identification and Characterization of a Gain-of-Function RAG-1 Mutant

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