Hairpin Formation in Tn5 Transposition

Abstract

The initial chemical steps in Tn5transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3′ hydrolytic nick by transposase. The free 3′OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3′ and 5′ strands without massive protein/DNA rearrangements. The initial chemical steps in Tn5transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3′ hydrolytic nick by transposase. The free 3′OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3′ and 5′ strands without massive protein/DNA rearrangements. base pair outside end inside end polyacrylamide gel electrophoresis Genomic DNA is known to undergo a variety of rearrangements, including inversions, deletions, duplications, and translocations. Transposition is one of the ways by which these rearrangements occur. The transposon Tn5 is a 5.8-kilobase pair prokaryotic, composite transposon consisting of two inverted repeats, IS50L and IS50R, that flank genes encoding antibiotic resistance. IS50R codes for the Tn5transposase (Tnp), the protein catalyzing all the steps in transposition. Each IS50 repeat is bracketed by two different 19-bp1 end sequences, termed the outside end (OE) and the inside end (IE), that are specifically recognized by Tn5 transposase (Fig. 1) (reviewed in Refs. 1Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington, D. C.1989: 185-210Google Scholar and 2Reznikoff W.S. Annu. Rev. Microbiol. 1993; 47: 945-963Crossref PubMed Google Scholar). Transposition of the full Tn5 element requires two OEs, whereas transposition of an IS50 element requires one OE and one IE.In vitro, Tn5 transposition requires only a hyperactive mutant transposase, such as EK54/LP372 Tnp, Mg2+, transposon DNA defined by two inverted 19-bp end sequences, and target DNA (3Goryshin I.Y. Reznikoff W.S. J. Biol. Chem. 1998; 273: 7367-7374Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Transposition frequency can be increased by using the mosaic end sequence (ME), a hyperactive, synthetic end sequence that is a hybrid of the OE and IE (Fig. 1) (4Zhou M. Bhasin A. Reznikoff W.S. J. Mol. Biol. 1998; 276: 913-925Crossref PubMed Scopus (58) Google Scholar). Tn5 is a member of the “cut and paste” family of transposons that includes Tn10 and Tn7. Although the transposition reactions vary in details, they follow the same basic mechanism. Transposase binds to the transposon DNA at the end recognition sequences. Then, the end sequences are brought together via transposase oligomerization to form a complex nucleoprotein structure, 2A. Bhasin, I. Y. Goryshin, M. Steiniger-White, D. York, and W. S. Reznikoff, manuscript in preparation. termed a synaptic complex (5Mizuuchi M. Baker T.A. Mizuuchi K. Cell. 1992; 70: 303-311Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 6Sakai J. Chalmers R.M. Kleckner N. EMBO J. 1995; 14: 4374-4383Crossref PubMed Scopus (95) Google Scholar). Once a stable synaptic complex has been formed, the transposon end sequence-donor DNA boundary can be cleaved to release a pair of 3′OHs at the ends, which are then used in the next chemical step, strand transfer (Fig. 2 A). Strand transfer occurs via a one step transesterification (7Mizuuchi K. Adzuma K. Cell. 1991; 66: 129-140Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 8Mizuuchi K. Annu. Rev. Biochem. 1992; 61: 1011-1051Crossref PubMed Scopus (314) Google Scholar) in which the 3′OHs attack phosphodiester bonds in the target DNA in a staggered fashion. For Tn5, the staggered attack leads to 9-bp gaps flanking the integrated transposon. The gaps are presumed to be filled in and ligated in the cell, and this leads to 9-bp duplications of the target sequence flanking the transposon (9Sasakawa C. Uno Y. Yoshikawa M. Mol. Gen. Genet. 1981; 182: 19-24Crossref PubMed Scopus (21) Google Scholar, 10Syvanen M. Hopkins J.D. Clements M. J. Mol. Biol. 1982; 158: 203-212Crossref PubMed Scopus (21) Google Scholar). In replicative transposition, the strand transfer product is a cointegrate, which is processed by DNA replication (11Mizuuchi K. Cell. 1983; 35: 785-794Abstract Full Text PDF PubMed Scopus (96) Google Scholar). The cleavage step differentiates cut and paste transposition from replicative transposition. In replicative transposons, such as phage Mu, only the 3′ end of the transposon is nicked to release a 3′OH, whereas the 5′ end remains covalently joined to donor DNA (7Mizuuchi K. Adzuma K. Cell. 1991; 66: 129-140Abstract Full Text PDF PubMed Scopus (138) Google Scholar). On the other hand, both the 3′ and 5′ ends of cut and paste transposons are cleaved from the flanking donor DNA. The Tn5 transposon is excised from the donor DNA by flush double strand breaks precisely at the end sequences (3Goryshin I.Y. Reznikoff W.S. J. Biol. Chem. 1998; 273: 7367-7374Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), as is Tn10 (12Morisato D. Kleckner N. Cell. 1984; 39: 181-190Abstract Full Text PDF PubMed Scopus (51) Google Scholar). Tn7 is excised with three-nucleotide 5′ overhangs (13Bainton R. Gamas P. Craig N.L. Cell. 1991; 65: 805-816Abstract Full Text PDF PubMed Scopus (117) Google Scholar). In this study, we examine how these double strand breaks arise. Previous work on Tn10 showed that the 3′ and 5′ nicks did not occur simultaneously, but rather were sequential with the 3′ nick preceding the 5′ nick (14Bolland S. Kleckner N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7814-7818Crossref PubMed Scopus (32) Google Scholar). In addition, in Mu, a single transposase active site carries out all the chemical steps (cleavage and strand transfer) at one end sequence (15Namgoong S.-Y. Harshey R. M. EMBO J. 1998; 17: 3775-3785Crossref PubMed Scopus (61) Google Scholar). Similarly, in Tn10, evidence suggests that a single transposase active site can also carry out all chemical steps at one end (16Bolland S. Kleckner N. Cell. 1996; 84: 223-233Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Like Tn10 transposase, Tn5transposase is a single polypeptide chain. It is difficult to imagine how a single active site can cleave both the 3′ and 5′ ends of a transposon without major conformational changes in the transposase and/or DNA. This is commonly known as the polarity problem. Tn7 has bypassed this problem by having its transposase made up of two polypeptides, TnsA and TnsB. TnsB contains the active site responsible for 3′ nicking, whereas TnsA contains the active site for 5′ nicking (17Sarnovsky R.J. May E.W. Craig N.L. EMBO J. 1996; 15: 6348-6361Crossref PubMed Scopus (125) Google Scholar). V(D)J recombination, an antigen receptor recombination system mechanistically similar to transposition, bypasses this polarity problem by going through a hairpin intermediate (18McBlane J.F. van Gent D.C. Ramsden D.A. Romeo C. Cuomo C.A. Gellert M. Oettinger M.A. Cell. 1995; 83: 387-395Abstract Full Text PDF PubMed Scopus (592) Google Scholar). It has recently been found that the Tn10 reaction also proceeds via a hairpin intermediate (19Kennedy A.K. Guhathakurta A. Kleckner N. Haniford D.B. Cell. 1998; 95: 125-134Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The model for cleavage via a hairpin is as follows. The first cleavage step is a 3′ hydrolytic nick by transposase at the end sequence. The free 3′OH attacks the 5′ end, forming a hairpin at the transposon end and releasing the flanking donor DNA. In order to undergo strand transfer, the hairpin needs to be resolved. The hairpin is hydrolytically cleaved by transposase in a step chemically identical to the first cleavage step, 3′ nicking. Cleavage of the hairpin frees a 3′OH. This 3′OH joins to target DNA in a strand transfer reaction chemically similar to the hairpin formation step (Fig. 2 B). Note that this mechanism utilizes only two repetitive chemistries, nicking and strand transfer. Furthermore, this model allows for a single transposase active site to perform all the necessary chemical steps without having to undergo a conformational the polarity problem. transposon in Tn5 cleavage we the cleavage step in Tn5 transposition. demonstrate that Tn5 via a hairpin intermediate. formation is for blunt end cleavage of the transposon. hairpins can be cleaved to form a transposon. hairpins can also be and Cleavage of the imprecise hairpin leads to precise 3′ end This of imprecise hairpin formation and on the transposon end is of the end sequence is for hairpin DNA were 5′ using and were in by at for and then the reaction at EK54/LP372 transposase using the protein system The contains the transposase to the through an It by (3Goryshin I.Y. Reznikoff W.S. J. Biol. Chem. 1998; 273: 7367-7374Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) with in with and with The pair transposase and ligated the of the were to an A of and were then with for at were and in of a were and the by a of of The with of with of and then at from the with and transposase in the first of The EK54/LP372 transposase to be at by and of by were at C. A 5′ used to for in the EK54/LP372 transposase and in a mutant transposase preparation. A. and W. S. Reznikoff, manuscript in preparation. A single base to of DNA for both protein not The used in of the in cleavage reactions A made by two mosaic end mosaic end is in transposon and donor and is a hairpin with two mosaic ends to The hairpin DNA from a polyacrylamide gel and to The strands of the DNA were also and The reactions were on a polyacrylamide A of the mosaic end DNA are In cleavage reactions of mosaic end DNA to and EK54/LP372 transposase, in a of were at for were by and in the of of were in and an of J. A Scholar) were then for and through polyacrylamide were and with a hairpin DNA from in cleavage reactions excised from polyacrylamide The excised DNA in (Fig. in (Fig. at The in the of and in This DNA then through a gel (Fig. to chemical (Fig. W. 65: PubMed Scopus Google Scholar). To the a the mosaic end the of the and a at the 5′ to a the mosaic end, the of the and a at the 3′ DNA to form the of this 5′ whereas with and in cleavage reaction with and were at and were and on a polyacrylamide The hairpins were from the gel and in were in the of and in The used by with and in with and with DNA is the of the but contains site by the of the The using the with and and the using a gel The hairpin ligated as a single strand the using DNA The and The DNA from by an and to the of and Reznikoff (3Goryshin I.Y. Reznikoff W.S. J. Biol. Chem. 1998; 273: 7367-7374Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). A in cleavage reaction consisting of mosaic end and EK54/LP372 transposase at were at and The reactions were by to a of and the on all of the were all were to and in the of were in for and through polyacrylamide were with a and precise hairpins were made by mosaic ends the of the the mosaic and a site as a 5′ The hairpin are The precise hairpin is and the imprecise hairpin is DNA were used as in an in were also used to strand transfer The precise imprecise hairpin with and The hairpins were then in with EK54/LP372 transposase and the as target DNA. The reaction were as with EK54/LP372 Tnp, of hairpin and of The reactions were at for 2 by transposition of two cleaved hairpins leads to of the The by of the two with DNA the of the transposon the of the Note that in these of the mosaic end from a to a to a This base on not The by and DNA from and For the we used and DNA The and the mosaic end in and were all 5′ A to the 3′ end of of the by The reaction of and of and at for The reaction at in the of and in The then with and This reaction of and of This reaction product the other were on a polyacrylamide gel for and by The were from the gel and in The were in the of and DNA were by The DNA were by of the and the of the and the whereas of the and the The of the were were then used in in cleavage reactions consisting of EK54/LP372 transposase, using A in Tn5 transposition is the blunt end cleavage of the transposon free from the donor DNA. We to the cleavage step in In this we the 19-bp mosaic end sequence with EK54/LP372 in a in the of The reactions are at to cleavage to and the cleavage are on polyacrylamide a we to two cleavage a transposon and a donor DNA. In addition to the we a at in a polyacrylamide gel (Fig. A). We that this a hairpin intermediate. To the a the DNA in the from a gel and on a polyacrylamide the DNA at base (Fig. B). This and is of a used to that this is a The hairpin DNA from a polyacrylamide gel and to chemical (Fig. W. 65: PubMed Scopus Google Scholar). The sequence strand DNA of the 3′ end of the mosaic end, and then the sequence to strand DNA at of the 5′ end of the mosaic end. that the of the on the hairpin sequence the of found in the strand sequence (Fig. and This sequence of to mosaic ends the hairpin sequence. The sequence difficult to of the 3′ end, that in the DNA. To the sequence of the hairpin we hairpin and to A DNA such that the hairpin a at the 5′ end, a at the 3′ end, and a sequence for in with this and the hairpin DNA from a polyacrylamide This hairpin DNA ligated as DNA which is the of the A the and for by resistance. the DNA be filled in by We and of the sequences for precise hairpins. hairpins of the 3′ end of the mosaic end covalently to of the 5′ end. This a hairpin of The other two from imprecise hairpins. In this the imprecise hairpins were from a of the 3′ end of the mosaic end and of the 5′ end. The imprecise hairpins a hairpin of (Fig. hairpins can also be on polyacrylamide We were in the hairpin is an intermediate in the Tn5 transposition reaction a A of an in cleavage reaction that the hairpin is an intermediate (Fig. A). The hairpin the first of the and to at this the hairpin of the the of hairpin in the reaction to at (Fig. The other in the reaction (Fig. B). The from to in The both nicked and double strand to of the at and then a DNA hairpin The donor DNA from precise hairpin formation and at at This product to at and then two hairpin and two donor DNA were The hairpin is an imprecise This at this made up of the The imprecise hairpin through the end of the imprecise hairpin a donor DNA This of the at It increased to of the at to other of were We that they are strand transfer by at in DNA. a of an in cleavage reaction showed that the hairpin is an we that hairpins be a for transposase cleavage. The hairpin were that a be precise and imprecise hairpin were 5′ used as in in and by polyacrylamide gel precise and imprecise hairpins can be cleaved at the 3′ as by release of the product (Fig. This of the hairpin is evidence that the hairpin is an intermediate in the cleavage The imprecise hairpin is cleaved the precise Although of the precise hairpins were only of the imprecise hairpins were We that the at in the precise hairpin reaction is a strand transfer It is that this not in the imprecise hairpin reaction cleavage of the imprecise hairpin is evidence that precise and imprecise hairpins can undergo strand transfer. imprecise hairpin strand transfer product has been and has the precise as as the 9-bp The base in the imprecise hairpin is not found in the strand transfer product not We that 5′ end cleavage requires formation of a hairpin on the transposon end and hairpin formation requires a 3′OH at of the strand to attack the To we mosaic end DNA that a a at of the In addition, we used a mosaic end DNA of the strand (Fig. A). hairpin formation requires a 3′OH, the be to hairpin formation and 5′ end cleavage. The with (Fig. as by for hairpin formation of and donor DNA release of A of the DNA the of the gel that a variety of the also (Fig. were of the with the with the with release also to only of the It that the 3′OH on 2 of the strand attacks the on the strand, as by release of the donor DNA and formation of the hairpin on this product we can that the 3′OH on 2 of the strand not attack 2 on the This result that the 3′ is for hairpin DNA this hairpin the did hairpin formation of (Fig. as as donor release of the The to a mosaic end by This reaction then with and and the polyacrylamide we not that the hairpin formation is a result of at of the We that EK54/LP372 transposase protein is with We that the the 3′ at in a of the this the is identical to the The is forming the hairpin in this The in the 5′ cleavage product as donor DNA with the the that hairpin formation is the mechanism for 5′ cleavage. In all the a This result from imprecise hairpin The and imprecise hairpins are in to the precise hairpins and not be on this The the of imprecise hairpins and in the be to in the and sequence of the donor The demonstrate that Tn5 via a DNA hairpin intermediate. in cleavage to the of a DNA gel and chemical and we that the is a hairpin at the transposon end sequences. We also that the hairpin is an intermediate in the transposition A showed the hairpin and then the of an intermediate. In addition, hairpins can be processed to blunt end transposons for strand transfer. formation is the mechanism for 5′ end as in the of this hairpin intermediate evidence for a model for the cleavage step in Tn5 transposition. The first step transposase-catalyzed 3′ hydrolytic nicks at of the end sequences to release a pair of 3′OHs at both ends of the transposon. free 3′OHs attack the phosphodiester bond and on the end sequences of the strands forming hairpins on the ends of the transposon DNA. The donor DNA is the hairpin formation This step is as as the 5′ cleavage In order for the transposon to its hairpin ends to be resolved. Transposase hydrolytically the hairpins in a step identical to the first chemical step releasing a pair of 3′OHs are of strand transfer the target a chemical step similar to hairpin In this the cleavage step is to two repetitive chemistries, nicking and strand transfer. This model is a mechanism by which one transposase active site can all the chemical reactions at one end without massive protein/DNA rearrangements. In addition, this mechanism also the mechanism of cleavage similar to that used by and (19Kennedy A.K. Guhathakurta A. Kleckner N. Haniford D.B. Cell. 1998; 95: 125-134Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, T.A. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). we found that imprecise hairpins are with precise hairpins. hairpins are hairpins that form the 3′OH on the strand attacks a other of the end sequence on the The imprecise hairpin we found is by the 3′OH at the phosphodiester bond and base the donor on the This leads to a hairpin with an base the end sequences. We also found that the imprecise hairpin to be resolved by transposase, the precise This suggests that transposase with a of the the by the the of transposase with the the of cleavage of the hairpin intermediate. We that the imprecise hairpin is resolved at a the precise This in the the precise hairpin resolved by whereas the imprecise hairpin (Fig. evidence that a resolved imprecise hairpin can undergo strand transfer. The strand transfer product has the 9-bp and the base is not the are a different that the the base be in the of the strand transfer It is that DNA in the gaps in the target DNA transposition (9Sasakawa C. Uno Y. Yoshikawa M. Mol. Gen. Genet. 1981; 182: 19-24Crossref PubMed Scopus (21) Google Scholar, A.K. Guhathakurta A. Kleckner N. Haniford D.B. Cell. 1998; 95: 125-134Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The of DNA the In addition, the base It is also that transposase is responsible for of the base to It is that the 3′OH hairpin cleavage attack the strand, but precisely this This attack to hairpin formation and of the 5′ single base This precise hairpin can be resolved and The that an imprecise hairpin must undergo a cleavage reaction to an of in the DNA ends are processed such that the DNA ends 5′ This the cleaved imprecise which has a 5′ It has been that the overhangs are by and/or by the DNA mechanism of 1992; PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google Scholar). the of the end sequence on the strand a of hairpins in (Fig. hairpins were with the with the It is that the 3′OH of 2 is of forming hairpins. This leads to the that the of not hairpin but is for hairpin This with V(D)J recombination In V(D)J recombination, the of the recognition sequence is to the DNA. and 2 of the are to and 2 of the Tn5 mosaic end sequence. at and 2 of the specifically hairpin and 2 are for hairpin formation D.A. J.F. van Gent D.C. Gellert M. EMBO J. 1996; 15: PubMed Scopus Google Scholar). Similarly, Tn10 suggests that at and 2 of the end sequence steps of the ends but full D. Kleckner N. EMBO J. PubMed Scopus Google Scholar). both and 2 of the Tn5 mosaic end sequence hairpin the 3′OH of 2 on the attacks DNA on the strand rather 2 phosphodiester in V(D)J recombination, imprecise nicks the occur. The 3′OHs from these nicks also attack at the D.A. J.F. van Gent D.C. Gellert M. EMBO J. 1996; 15: PubMed Scopus Google Scholar). transposase and the can a 3′OH in active site in such a as to perform the This at in the DNA and/or The of Tn5 transposase to cleave hairpin DNA that transposase binds hairpin DNA in addition to end sequence DNA the initial step in transposase binds these two different DNA is that transposase a conformational in order to with the hairpin and this conformational with the of the It is that transposition, transposase and the DNA are conformational which to a variety of nucleoprotein of transposase to two end sequence the of form the first order nucleoprotein in Tn5 transposition, the synaptic Transposase to hairpin DNA is a order nucleoprotein different the synaptic a intermediate The of transposition nucleoprotein is not a In transposition, stable nucleoprotein been M.A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Tn5transposition also proceeds through complex The of hairpins in transposition and is a in transposition. Tn10 found to via a hairpin intermediate (19Kennedy A.K. Guhathakurta A. Kleckner N. Haniford D.B. Cell. 1998; 95: 125-134Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). proceeds by a mechanism similar to hairpin formation but a intermediate In this a 3′ nick is made at one transposon end. The 3′OH attacks the opposite transposon end a on the same strand, forming a The strand has donor DNA this for strand transfer P. M. EMBO J. 1998; 17: PubMed Scopus Google Scholar). In Tn5, a intermediate not but rather hairpins are on both transposon ends of different transposon with transposase and for different hairpins, we found that the 3′OH attacks the opposite phosphodiester bond on its end sequence rather on the end sequence Bhasin, I. Y. Goryshin, M. Steiniger-White, D. York, and W. S. Reznikoff, manuscript in preparation. The V(D)J recombination system is chemically similar to transposition. In the V(D)J recombination and been to carry out transposition A. 1998; PubMed Scopus Google Scholar, K. M. Gellert M. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). V(D)J recombination also proceeds via a hairpin hairpins are on the DNA to donor and not on the recognition sequence DNA to transposon In V(D)J recombination, a 5′ nick is made to the recognition sequence. The free 3′OH on the DNA end a attack on the strand of the DNA releasing the recognition sequence. In this the attack occurs on the phosphodiester bond opposite (18McBlane J.F. van Gent D.C. Ramsden D.A. Romeo C. Cuomo C.A. Gellert M. Oettinger M.A. Cell. 1995; 83: 387-395Abstract Full Text PDF PubMed Scopus (592) Google Scholar). In a family transposon from the a hairpin mechanism is to the transposition. The hairpin mechanism is identical to V(D)J 5′ nicks are made at the phosphodiester bond one base outside the transposon. the 3′OH not attack the phosphodiester bond but rather attacks the phosphodiester bond 5′ to Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). This is the same as the imprecise hairpin formation that we found in transposons a hairpin mechanism that the transposase not to undergo major conformational changes in order to perform a variety of chemical We Steiniger-White, and for and for We also and for work on the