RAG1/2-Mediated Resolution of Transposition Intermediates

Melek, Meni; Gellert, Martin

doi:10.1016/s0092-8674(00)80874-0

Cited by 65 publications

(92 citation statements)

References 34 publications

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“…The formation of DNA hairpins is physiologically the most important DNA strand transfer reaction that the RAG proteins catalyze. However, they are also known to mediate several alternative DNA strand transfer reactions, including the rejoining of signal ends and coding ends in either the original or the opposing configuration in a reversal of the cleavage reaction (yielding open-shut joints or hybrid joints, respectively) (27,35), the integration of signal ends into nonhomologous DNA by classical transposition (1,17), a retrovirus-like disintegration reaction that reverses transpositional strand transfer (34), and an "inverse transposition" reaction, in which a non-RSS DNA strand is transferred into a canonical RSS (48). In principle, one might expect that since the RAG-1 E649A mutation promotes accelerated hairpin formation, this RAG-1 mutant might similarly exhibit enhanced ability to mediate alternative DNA strand transfer reactions.…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

“…We next considered whether the RAG-1 E649A mutation enhances a disintegration reaction that models the reversal of transpositional strand transfer (34). For these experiments, a substrate containing a 12-RSS integrated into a radiolabeled target DNA was incubated with the RAG proteins in the presence of either Mg 2ϩ or Ca 2ϩ .…”

Section: Resultsmentioning

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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“…Intact substrates were used for transposition into 100 ng of pBR322 by using 5 mM MgCl 2 to catalyze the reaction (44).…”

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

confidence: 99%

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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“…(67) However, no examples of transpositional insertion of DNA (bounded by signal ends) at chromosomal translocation sites has ever been reported in tumors. Nevertheless, there are hit-and-run mechanisms that one can speculate could occur such that the signal ends transpose in, but then disengage (called transpositional disintegration (84) ), resulting in a DSB). Such events should leave behind a hairpin at the site of disintegration and the necessary opening of those hairpins should leave behind short inverted repeats called P nucleotides (P stands for palindrome).…”

Section: Does Rag-mediated Transposition Lead To Chromosomal Translocmentioning

confidence: 99%

DNA structures at chromosomal translocation sites

Raghavan

Lieber

2006

BioEssays

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SummaryIt has been unclear why certain defined DNA regions are consistently sites of chromosomal translocations. Some of these are simply sequences of recognition by endogenous recombination enzymes, but most are not. Recent progress indicates that some of the most common fragile sites in human neoplasm assume non-B DNA structures, namely deviations from the Watson-Crick helix. Because of the single strandedness within these non-B structures, they are vulnerable to structure-specific nucleases. Here we summarize these findings and integrate them with other recent data for non-B structures at sites of consistent constitutional chromosomal translocations.

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RAG1/2-Mediated Resolution of Transposition Intermediates

Cited by 65 publications

References 34 publications

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

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

Requirements for DNA hairpin formation by RAG1/2

DNA structures at chromosomal translocation sites

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