Tn10 Transposition via a DNA Hairpin Intermediate

Kennedy, Angela K; Guhathakurta, Anjan; Kleckner, Nancy; Haniford, David B.

doi:10.1016/s0092-8674(00)81788-2

Cited by 192 publications

(160 citation statements)

References 30 publications

(20 reference statements)

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“…One potential difference between these two types of transpososomes may be the trigger for the conformational change. Cut-and-paste transposases, such as Tn10 and Tn5 (40,41), also catalyze cleavage of the non-transferred strand at each transposon DNA end. This cleavage occurs via a two-step process in which the 3ЈOH of the cleaved transferred strand at the transposon end directly attacks the non-transferred strand to generate a DNA hairpin intermediate.…”

Section: Discussionmentioning

confidence: 99%

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Williams¹,

Baker

2004

Journal of Biological Chemistry

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Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 -flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 -flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.The successful relocation of mobile genetic elements, as with all DNA rearrangement processes, requires the precise spatial and temporal organization of DNA components within the active sites of large nucleoprotein complexes. This dynamic organization permits the proper series of DNA cleavage and joining reactions that comprise each DNA recombination pathway. Transposition and retroviral integration occur via one of two pathways that share common reaction steps (1, 2). Similar phosphoryl transfer reactions also take place during the early steps of VDJ recombination (3). Many transposases and integrases form a recombinase family related both by the threedimensional structure of their catalytic domains and by a conserved set of acidic active site residues (4). Bacteriophage Mu transposase is a well studied member of this family.Transposases and integrases promote recombination via either a replicative or cut-and-paste transposition pathway. These pathways share two common steps (see Fig. 1). First, one strand at each element end is hydrolyzed to yield a 3ЈOH at the terminus of the element sequence. This donor cleavage step separates this strand at each element end from the surrounding (or flanking) DNA. In the second common step, called DNA strand transfer or joining, each liberated 3ЈOH directly attacks closely spaced phosphodiester bonds in opposite strands of the DNA at the new location (the target DNA). Mu transposase promotes these two steps during replicative transposition to yield a transposition product in which each transposon end is covalently attached ...

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Section: Discussionmentioning

confidence: 99%

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Williams¹,

Baker

2004

Journal of Biological Chemistry

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“…Similar findings were made with mutagenesis of equivalent residues in Tn10 (W265) (21) and Hermes (W319) transposases (22). Thus, parallel mechanisms may operate in transposases in which the hairpin is on the transposon end (Tn5 and Tn10), and those in which the hairpin is on the flanking DNA (Hermes and RAG1/2) (18,23). In this work, we used abasic substrates to investigate the role of bases around the coding-RSS border during the two steps of DNA cleavage.…”

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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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“…The process of cut-and-paste transposition by the bacterial elements Tn10 (25) and Tn5 (26) has been shown to involve the formation of a hairpin intermediate. Structural analysis of the Tn5 transposase (27,28) coupled with biochemical and genetic experiments on both Tn5 (29,30) and Tn10 (31) has defined a hairpin binding module in these transposases (32).…”

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confidence: 99%

Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

Bankhead

Chaconas

2004

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

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The ResT protein, a telomere resolvase from Borrelia burgdorferi, processes replication intermediates into linear replicons with hairpin ends by using a catalytic mechanism similar to that for tyrosine recombinases and type IB topoisomerases. We have identified in ResT a hairpin binding region typically found in cut-and-paste transposases. We show that substitution of residues within this region results in a decreased ability of these mutants to catalyze telomere resolution. However, the mutants are capable of resolving heteroduplex DNA substrates designed to allow spontaneous destabilization and prehairpin formation. These findings support the existence of a hairpin binding region in ResT, the only known occurrence outside a transposase. The combination of transposaselike and tyrosine-recombinase-like domains found in ResT indicates the use of a composite active site and helps explain the unique breakage-and-reunion reaction observed with this protein. Comparison of the ResT sequence with other known telomere resolvases suggests that a hairpin binding motif is a common feature in this class of enzyme; the sequence motif also appears in the RAG recombinases. Finally, our data support a mechanism of action whereby ResT induces prehairpin formation before the DNA cleavage step.

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Tn10 Transposition via a DNA Hairpin Intermediate

Cited by 192 publications

References 30 publications

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Requirements for DNA hairpin formation by RAG1/2

Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

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