 |
The Mechanism and Regulation of Transposition Reactions
Summary: Nancy Craig's work is focused on the molecular mechanism by which a transposable element moves. 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 is 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 transposons. One element, the bacterial transposon Tn7, displays unusual target selectivity. Most transposable elements display limited site selectivity, inserting into many different target sites. By contrast, Tn7 inserts into a single specific site, called attTn7. When attTn7 is unavailable, Tn7 inserts at low frequency into many different sites. The other elements we study are members of the eukaryotic hAT and piggyBac families. There are hAT elements that can transpose in vertebrates and intact hAT transposase-like proteins in mammalian genomes, including the human genome. There are piggyBac elements in animal genomes and piggyBac transposase-like proteins in many genomes, including the human genome.
We began our work on hAT elements with Hermes, an element closely related to the hobo element of Drosophila. We also study AeBuster1, the first active transposon to be found in the mosquito Aedes aegypti, the vector of malaria, and Tol2, a medaka fish element that can also transpose in zebrafish.
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. Host proteins, such as DNA-bending proteins, may also be involved in transposition in vivo.
We are interested in understanding interactions between mobile elements and their hosts, such as how the host may regulate 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.
Further studies involve using transposons as tools for genome engineering. For example, 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 property is an attractive one for making bacterial strains for vaccine production. Also, there are orthologs of attTn7 in the human genome. We are asking if a Tn7-based system can be used in metazoans to make highly site-specific insertions, a very desirable property for gene therapy. We are also interested in using hAT and piggyBac elements as tools for the engineering of mammalian genomes. Other researchers previously established that Tol2 can transpose in mammalian cells, and we have recently established that several other hAT elements can also transpose in mammalian cells. To facilitate their use for the engineering of mammalian genomes, we are making derivatives of these transposases that promote transposition at higher frequencies, and we are exploring adding sequence-specific DNA-binding domains to such transposases to direct site-specific insertion into mammalian genomes.
hAT Elements
We are studying the recombination of Hermes in vitro to dissect its 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. We are focusing on determining how the gap that results from Hermes excision from the donor site is repaired and how host factors may influence Hermes transposition and the repair of the broken DNAs that occur during transposition. We have used this yeast system to isolate Hermes mutants that transpose at higher than wild-type frequency, and we will apply this strategy to the other hAT elements. Understanding how changes in protein sequence can lead to increased transposition will help us understand the structure and function of the Hermes transposase. Such increased frequency of transposition mutants will also have applications for genome engineering. We expect that the isolation of AeBuster1 high-frequency transposition mutants will be valuable for genome engineering in Ae. aegypti. We are also using the yeast system to analyze the TcBuster and Tol2 transposition systems. We will look for transposition host factors and DNA repair following transposition in the natural hosts of these elements, for example, for Hermes in Drosophila and Tol2 in zebrafish.
Transposons with a transposase like that of AeBuster1 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.
piggyBac
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; we and others previously showed that hAT, Tc1/mariner, the bacteriophage Mu, and other bacterial elements, such as Tn5 and Tn10 transposases and retroviral integrases, are members of the DDE family of recombinases. We have now shown that piggyBac is also a member of this family.
Tn7
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.
The regulation of Tn7 transposition by host factors and the repair reactions that are required to generate intact duplex DNA from the actual products of transposition are also of interest to us.
Our 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. Our work on AeBuster1 is supported by the Johns Hopkins Malaria Research Institute.
Last updated: July 9, 2007
|
|
|
|
