Hairpin Formation in Tn5 Transposition

Bhasin, Archna; Goryshin, Igor Y.; Reznikoff, William S.

doi:10.1074/jbc.274.52.37021

Cited by 145 publications

(129 citation statements)

References 29 publications

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“…Recombinases, integrases, transposases, and topoisomerases have all been shown previously to catalyze DNA strand transfer reactions (8,37,(39)(40)(41)(42). We have identified here a restriction enzyme that cannot only cleave DNA but can also catalyze strand transfer reactions, which possibly implies novel functions for these enzymes.…”

Section: Discussionmentioning

confidence: 99%

Site-specific DNA transesterification catalyzed by a restriction enzyme

Sasnauskas¹,

Connolly

Halford

et al. 2007

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

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Most restriction endonucleases use Mg 2؉ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5 terminus of the cleaved DNA. Under certain conditions, the terminal 3 -OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a twostep mechanism. We propose that BfiI first makes a covalent enzyme-DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively.I n the presence of Mg 2ϩ , type II restriction endonucleases (REases) hydrolyze specific phosphodiester bonds in DNA to yield 5Ј-phosphate and 3Ј-hydroxyl termini (1). The stereochemical pathway for phosphodiester hydrolysis by four such enzymes, EcoRI, EcoRV, SfiI, and HpaII (2-4), have been determined. All proceed with inversion of configuration at the scissile phosphate, which implies an odd number of chemical steps in the hydrolytic reaction, and it is most simply accounted for by a direct in-line displacement of the 3Ј-leaving group by water (5-7). High-resolution structures are available for many more REases than have been analyzed stereochemically (1). Their active sites are generally similar, with several carboxylates to act as ligands for Mg 2ϩ in spatially equivalent positions (1). All such enzymes probably act in the same way, by using water to attack the scissile phosphate, to invert its configuration.Endonucleases from the transposase-retroviral integrase family, like MuA or HIV-integrase, catalyze at a single active site two sets of reactions on DNA (4, 8, 9). The first, the endonucleolytic cleavage of the 3Ј end of the transposon or viral DNA, is mechanistically similar to the hydrolysis reactions of REases. The second, termed DNA strand transfer, is a single-step transesterification in which the newly liberated 3Ј-OH group attacks and becomes linked to a phosphate group in the target DNA (9, 10).We ask here whether the BfiI REase can catalyze transesterification reactions in addition to DNA hydrolysis. BfiI cleaves DNA at specified positions downstream from an asymmetric recognition sequence (11). Unlike other REases, it functions without metal ions (12). The N-terminal half of BfiI (13, 14) is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium (15, 16) that belongs to the phospholipase D (PLD) superfamily. The PLD superfamily is a diverse group of proteins that includes phospholipases, phospholipid synthases, bacterial toxins, phosphodiesterases (PDEs), and nucleases (17, 18). The PLD enzymes catal...

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Section: Discussionmentioning

confidence: 99%

Site-specific DNA transesterification catalyzed by a restriction enzyme

Sasnauskas¹,

Connolly

Halford

et al. 2007

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

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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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“…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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Hairpin Formation in Tn5 Transposition

Cited by 145 publications

References 29 publications

Site-specific DNA transesterification catalyzed by a restriction enzyme

Site-specific DNA transesterification catalyzed by a restriction enzyme

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

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

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