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The Mechanism and Regulation of DNA Transposition Reactions
Summary: Nancy Craig's work is focused on the molecular mechanisms by which transposable elements move and how they can be exploited for genome engineering. These elements are discrete pieces of DNA that can move between many different insertion sites. They are present in virtually all organisms and contribute to both genome structure and function.
Despite the essential role of DNA in maintaining accurate genetic information, this molecule displays a surprising degree of plasticity. DNA rearrangements—the reorganization of DNA sequences by breakage, translocation, and rejoining reactions—mediate a wide variety of fundamental cellular processes. These rearrangements play an important role in the acquisition of new genetic elements such as viruses, in the control of gene expression during development, and in the repair of damaged DNA. We are particularly interested in the type of recombination called transposition, in which a discrete DNA segment moves from one donor position and inserts into another, nonhomologous target site.
Transposable elements have been identified in virtually all organisms. Strikingly, about half the human genome and most mammalian genomes are composed of DNA sequences related to these elements, which can have profound effects on genome structure and function. For example, transposon insertion into a gene will likely inactivate that gene; transposon insertion into DNA sequences that control the expression of nearby genes may inactivate or activate those genes. Indeed, the movement of transposable elements has resulted in a number of human diseases.
Our research is focused on several different DNA cut-and-paste transposons that move by excision from the donor site by double-strand breaks at each end of the element, followed by joining of these newly exposed transposon ends to a new insertion site. Most such transposable elements display limited site selectivity, inserting into many different target sites. One element that we study, however, the bacterial transposon Tn7, displays unusual target selectivity, inserting at high frequency into a single specific site, called attTn7.
The other elements we study are members of the widespread eukaryotic hAT and piggyBac superfamilies; there are active members of these families in vertebrates. We were originally attracted to the study of these elements because nothing was known about their transposition at the molecular level. Many genomes, including the human genome, also contain intact hAT and piggyBac transposase-like proteins whose functions interest us.
We are studying several hAT elements: Hermes, an element closely related to the hobo element of Drosophila; AeBuster1, the first active transposon to be found in the mosquito Aedes aegypti, the vector of malaria; TcBuster, an element from the red beetle Tribolium castaneum that we have found is highly active in mammalian cells; and Tol2, a medaka fish element that can also transpose in zebrafish and mammalian cells.
We are studying Trichoplusia ni piggyBac from the cabbage looper moth, which has been used for transgenesis in many other organisms, and Myotis lucifugus piggyBac, an element from the little brown bat. Notably, we have identified M. lucifugus piggyBac as the first currently active DNA transposon in a mammalian genome. Previous studies found no evidence that DNA cut-and-paste elements have been active in the past 35 million years in mammalian genomes.
We are interested in understanding in molecular detail how these elements move from place to place and how the frequency of this movement is modulated. Like most mobile DNA segments, the Tn7, hAT, and piggyBac elements encode their own transposition machinery, consisting of recombination proteins that act on special sequences at the termini of the element to excise the element from its donor site and join these termini to the new insertion site. To probe the transposition mechanisms of the elements, we have developed in vitro systems reconstituted with purified proteins. We have found that common chemical mechanisms underlie the movement of all of these elements and that the active-site regions of their transposases are actually intimately related in structure. The similarities between these elements reflect the fact that the breakage and joining reactions that underlie the movement of all DNA cut-and-paste elements and the DNA forms of retroviruses are highly similar and that their transposases and integrases are structurally related.
Other studies in our lab involve developing transposons as tools for genome analysis and engineering in bacteria, yeast, and mammalian cells.
hAT Transposition Systems
We are studying the recombination of hAT elements Hermes, AeBuster1, TcBuster, and Tol2 in vitro to dissect the hAT transposition mechanism. The reactions that excise Hermes from its donor site are identical to the reactions used in V(D)J recombination to initiate the assembly of the coding regions of the proteins of the immunoglobulin superfamily from different gene segments. In our in vitro studies, we are dissecting the mechanism of DNA-strand breakage and joining through site-directed mutagenesis and examining alternative DNA substrates to probe the roles of particular amino acids in the active site in recombination. We have also developed and are dissecting in vitro systems for AeBuster1, TcBuster, and Tol2.
The development of a Hermes transposition system in Saccharomyces cerevisiae allows us to use genetic methods to probe this transposition system, and we are applying this system to the other hAT elements. One aspect of our work is isolating hyperactive versions of transposable elements. Understanding how and why these elements are hyperactive will help us to understand mechanism and control of transposition at a higher resolution.
piggyBac Transposition Systems
Using an in vitro transposition system, we have determined the mechanism of piggyBac transposition. We find that piggyBac excises from the donor DNA by double-strand breaks that involve the transient formation of DNA hairpins on the transposon end. It then inserts into the target DNA by attack of the 3'OH transposon ends on the target DNA. This mechanism, using direct phosphoryl transfer reactions, is the same used by the hAT, Tn7, and other transposition systems that use DNA intermediates, such as retroviral integrases. This unity in mechanism reflects a unity in the structure of the catalytic domains of the transposases and integrases that promote these reactions.
The piggyBac transposition mechanism also explains several other features of piggyBac transposition, notably its exclusive insertion into TTAA target sites and its precise excision from these sites. As with hAT elements, we have developed piggyBac transposition systems in S. cerevisiae and are using these to make hyperactive transposase derivatives.
The Tn7 Transposition System
In contrast to the single transposition protein encoded by hAT and piggyBac elements, Tn7 encodes five transposition proteins (TnsA, TnsB, TnsC, TnsD, TnsE). These five Tns proteins promote two distinct but overlapping transposition pathways that differ in their target sites and in their target site–selecting protein, TnsD or TnsE. TnsABC are necessary but not sufficient for all transposition reactions. In the presence of TnsD (i.e., with TnsABC+D), Tn7 integrates into attTn7; in the presence of TnsABC+E, Tn7 preferentially inserts into certain plasmid replicons that can move between cells, thus promoting the spreading of Tn7 from organism to organism, a process important in the rapid dissemination of antibiotic resistance among bacterial populations.
Two Tns proteins, TnsA and TnsB, act together to form the transposase, i.e., the recombinase that specifically recognizes the special sequences at the ends of the transposon and executes the breakage and joining reactions that underlie transposition. Both TnsA and TnsB play direct roles in the breakage and joining reactions: each of these proteins contains an active site for a DNA-processing reaction. TnsB is a member of the retroviral integrase superfamily of transposases, whereas TnsA is related to restriction enzymes.
The transposase TnsAB is not constitutively active; TnsAB activity is modulated and directed to particular target sites by the other Tns proteins. The central regulator in this process is TnsC, which interacts with both TnsA and TnsB to activate the transposase activity and also plays a key role in target site selection by interacting with the target DNA and a targeting protein, TnsD or TnsE. TnsD interaction with attTn7 generates an altered DNA structure that is recognized by TnsC.
We have shown that the breakage and joining steps of Tn7 transposition to attTn7 initiate only when a nucleoprotein complex forms containing the DNA substrates of transposition, i.e., the transposon ends and the attTn7 target DNA, and the Tns proteins. We are working to define the elaborate network of protein-DNA and protein interfaces involved in this complex, to understand how it is assembled, and to decipher how these interactions activate the catalytic sites of TnsAB.
We have used genetic strategies to isolate interesting variants of the Tns proteins that have deepened our understanding of Tn7 transposition control.
Cellular Control of and Responses to Transposition
We are also interested in understanding interactions between mobile elements and their hosts, for example, how the host may regulate transposition, how intact duplex DNA is generated from the primary products of transposition, and what impact transposition has upon the host. Since transposition involves the breakage of DNA molecules, host DNA repair systems are also intimately involved in transposition. In particular, we are using transposition systems for hAT and piggyBac elements that we have developed in the highly genetically tractable organism S. cerevisiae to probe these questions.
Using Transposons for Genome Analysis and Engineering
Transposons are powerful tools for insertional mutagenesis of many organisms and for the stable introduction of new DNA into a host by transgenesis. We are interrogating the organization of the DNA genomes into chromatin by analyzing high-resolution insertion site profiles of our transposons into the yeast and human genomes.
The hyperactive transposons we are isolating and analyzing will contribute to genome dissection in multiple organisms. For example, we have isolated hyperactive versions of the mosquito element AeBuster that may facilitate the now difficult engineering of many insect genomes. Related hyperactive TcBuster elements show increased activity in mammalian genomes. Our hyperactive piggyBac screens in yeast will also likely identify hyperactive mutants that will be useful in mammalian cell mutagenesis.
We have developed a Tn7-based system that mediates site-specific insertions into bacterial genomes so efficiently that no selection for the insertion event is required. This is an attractive property for making bacterial strains for vaccine production. Another significant advance in using transposable elements for mammalian cell transgenesis would be to develop transposons that will insert at specific sites so as to avoid unwanted mutagenesis of host genes. We are exploring adding sequence-specific DNA-binding domains to hAT and piggyBac transposases to direct site-specific insertion into mammalian genomes. Also, there are orthologs of attTn7 in the human genome that Tn7 inserts efficiently into in vitro, and we are interested in exploiting these sites for element insertion in vivo.
Domesticated Transposases
Transposons with a transposase like that of AeBuster1, other hAT elements, and piggyBac are widely distributed. They also are closely related, however, to proteins found in the human genome and in some other mammalian genomes. These mammalian Buster proteins do not appear to be part of transposable elements. These proteins are extremely similar in sequence, however, strongly suggesting they have been subject to purifying selection and thus provide important cellular functions. It is possible that they are "domesticated" transposases whose ability to bind DNA has been harnessed for some other cellular process—for example, the regulation of transcription—but which have lost the ability to promote DNA breakage and joining. We plan to explore the cellular roles of these proteins.
The work on host factors in both Hermes and Tn7 transposition and on Hermes high-frequency transposition mutants is supported by the National Institutes of Health. The work on AeBuster1 is supported by the National Institutes of Health and has received support from the Johns Hopkins Malaria Research Institute. The work on targeting transposon insertion in the human genome is sponsored by the Maryland Stem Cell Research Fund.
Last updated December 16, 2008
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