Complete transposition requires four active monomers in the mu transposase tetramer.

Baker, Tania A.; Kremenstova, E.; Luo, Li

doi:10.1101/gad.8.20.2416

Cited by 68 publications

(15 citation statements)

References 50 publications

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“…In contrast, a tetramer of MuA transposase efficiently integrates two synapsed transposon ends in in vitro transposition reactions (25,29). Single-end integration of Mu DNA, however, can be enhanced by using a mixture of wildtype and transposition-defective mutant proteins that carry amino acid substitutions in the active site of MuA (3). Whereas the level of single-end transposition product relative to that of the normal double-end product was increased by increasing the level of active-site mutant protein in the reaction mixture, mixtures of two different active-site mutants were inactive (3).…”

Section: Resultsmentioning

confidence: 99%

Asymmetric Processing of Human Immunodeficiency Virus Type 1 cDNA In Vivo: Implications for Functional End Coupling during the Chemical Steps of DNA Transposition

Chen

Engelman

2001

Molecular and Cellular Biology

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Retroviral integration, like all forms of DNA transposition, proceeds through a series of DNA cutting and joining reactions. During transposition, the 3 ends of linear transposon or donor DNA are joined to the 5 phosphates of a double-stranded cut in target DNA. Single-end transposition must be avoided in vivo because such aberrant DNA products would be unstable and the transposon would therefore risk being lost from the cell. To avoid suicidal single-end integration, transposons link the activity of their transposase protein to the combined functionalities of both donor DNA ends. Although previous work suggested that this critical coupling between transposase activity and DNA ends occurred before the initial hydrolysis step of retroviral integration, work in the related Tn10 and V(D)J recombination systems had shown that end coupling regulated transposase activity after the initial hydrolysis step of DNA transposition. Here, we show that integrase efficiently hydrolyzed just the wild-type end of two different single-end mutants of human immunodeficiency virus type 1 in vivo, which, in contrast to previous results, proves that two functional DNA ends are not required to activate integrase's initial hydrolysis activity. Furthermore, despite containing bound protein at their processed DNA ends, these mutant viruses did not efficiently integrate their singly cleaved wild-type end into target DNA in vitro. By comparing our results to those of related DNA recombination systems, we propose the universal model that end coupling regulates transposase activity after the first chemical step of DNA transposition.Transposition is a specialized type of DNA recombination that results in the movement of transposon or donor DNA from a preexisting genomic location to a new DNA target site. Transposition can be nonreplicative, wherein the transposon leaves its old location for the new site, or replicative, when a copy of the donor DNA is retained at the original genomic position. Examples of nonreplicative and replicative prokaryotic transposons are Tn10 and bacteriophage Mu, respectively. V(D)J recombination and retroviral integration represent nonreplicative and replicative eukaryotic transposition systems, respectively. Retroviruses in this sense are packaged RNA intermediates of replicative DNA transposition.Certain mechanistic features group these seemingly disparate elements into the same class of DNA recombination. Most notably, transposition proceeds through a common DNA recombination intermediate. This intermediate is formed by transposase-mediated intermolecular strand transfer of donor DNA 3Ј ends to the 5Ј-phosphates of a double-stranded staggered cut in target DNA (Fig. 1). In the intermediate, the 5Ј ends of donor DNA remain unjoined to the target. Subsequent DNA repair or replication enzymes fill in and ligate the singlestrand gaps, yielding the sequence duplication of the target DNA cut flanking the newly integrated element.Transposing through a higher-order nucleoprotein complex with a multimer of transposase eng...

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Section: Resultsmentioning

confidence: 99%

Asymmetric Processing of Human Immunodeficiency Virus Type 1 cDNA In Vivo: Implications for Functional End Coupling during the Chemical Steps of DNA Transposition

Chen

Engelman

2001

Molecular and Cellular Biology

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“…8 were seen previously using full-length protein (23). MuA, MuA-(77-663) (24) and MuB (25) were prepared as described.…”

Section: Methodsmentioning

confidence: 99%

“…The activity of transpososomes containing both wild-type and mutant subunits depends on the placement of subunits. For example, some mixed complexes are fully active, some are not active at all, and some are able to complete cleavage and strand transfer of only one of the two Mu DNA ends (23,29,30).…”

Section: Figmentioning

confidence: 99%

DNA Recognition Sites Activate MuA Transposase to Perform Transposition of Non-Mu DNA

Goldhaber-Gordon¹,

Williams²,

Baker³

2002

Journal of Biological Chemistry

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Mu transposition occurs within a large protein-DNA complex called a transpososome. This stable complex includes four subunits of MuA transposase, each contacting a 22-base pair recognition site located near an end of the transposon DNA. These MuA recognition sites are critical for assembling the transpososome. Here we report that when concentrations of Mu DNA are limited, the MuA recognition sites permit assembly of transpososomes in which non-Mu DNA substitutes for some of the Mu sequences. These "hybrid" transpososomes are stable to competitor DNA, actively transpose the non-Mu DNA, and produce transposition products that had been previously observed but not explained. The strongest activator of non-Mu transposition is a DNA fragment containing two MuA recognition sites and no cleavage site, but a shorter fragment with just one recognition site is sufficient. Based on our results, we propose that MuA recognition sites drive assembly of functional transpososomes in two complementary ways. Multiple recognition sites help physically position MuA subunits in the transpososome plus each individual site allosterically activates transposase.Transposons are found in all the biological kingdoms, and some perform specialized functions. For example, the machinery that initiates V(D)J recombination likely evolved from a transposon (1, 2), and the cDNA of HIV and other retroviruses integrate into host cell DNA through mechanisms nearly identical to transposition (3). The genome of bacteriophage Mu is a transposon that uses transposition both to integrate into the DNA of a new host cell and to replicate before lysis. Like most DNA rearrangements, transposition is a complex, multi-step process, requiring numerous DNA sequence elements. Studies of bacteriophage Mu have been central to our understanding of both the fundamental mechanisms and the complexities of DNA transposition.Phage Mu encodes a transposase, MuA, that transfers the Mu genome from one DNA location (the transposition donor) to a new location (the transposition target) (4, 5). During transposition, transposase performs two principle reactions: DNA cleavage and DNA strand transfer. During cleavage, the donor DNA is nicked twice, once at each 3Ј end of the Mu genome. During strand transfer, the cleaved transposon ends are inserted into neighboring sites on the two target strands.Little or no specific sequence information is needed on the target DNA (6), but the Mu DNA provides many sequence cues for transposition (Fig. 1). For example, the last two nucleotides at either 3Ј end of the Mu DNA, the cleavage sites, have the sequence 5Ј-CA. Also near each end of the Mu DNA are three recognition sites, distinct from the cleavage sites, which share a 22-base pair consensus sequence. The recognition sites are referred to as R1, R2, and R3 on the right end and L1, L2, and L3 on the left end ( Fig.

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“…This is reminiscent of recent findings on transposition by bacteriophage Mu. The MuA transposase protein, which carries out reactions related to those of retroviral integration, remains tightly associated with transposition intermediates (3,4,45). MuA must be removed by action of the ClpX protein chaperone in an ATP-dependent fashion to permit completion of Mu transposition (31,32).…”

Section: Discussionmentioning

confidence: 99%

Repair of Gaps in Retroviral DNA Integration Intermediates

Yoder

Bushman

2000

J Virol

180

165

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Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3 DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5 two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.

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Complete transposition requires four active monomers in the mu transposase tetramer.

Cited by 68 publications

References 50 publications

Asymmetric Processing of Human Immunodeficiency Virus Type 1 cDNA In Vivo: Implications for Functional End Coupling during the Chemical Steps of DNA Transposition

Asymmetric Processing of Human Immunodeficiency Virus Type 1 cDNA In Vivo: Implications for Functional End Coupling during the Chemical Steps of DNA Transposition

DNA Recognition Sites Activate MuA Transposase to Perform Transposition of Non-Mu DNA

Repair of Gaps in Retroviral DNA Integration Intermediates

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