The research in the laboratory is aimed at providing a mechanistic understanding of the control of the biological activity of macromolecular complexes and assemblies using structural approaches such as X-ray crystallography and cryo-electron microscopy as a principal tool. In particular, our research focuses on understanding the genome rearrangements that occur when discrete segments of DNA are moved around within cells. For example, DNA transposons reside in host cell genomes and can be moved (or copied) into new locations by self-encoded enzymes, and we are studying how these enzymes function. DNA can also originate from outside the cell - as in the case of DNA viruses - and we would like to understand how integrating viruses infect cells.

Among the simplest transposition systems are those of prokaryotic insertion sequences (IS). We have been investigating one particular IS, ISHp608, originally isolated from the pathogenic bacterium, Helicobacter pylori. The transposase which moves ISHp608, known as TnpA, has only 155 amino acids and is among the smallest of all known transposases. We used a combined structural and biochemical approach to show that this transposase uses a completely novel mechanism for moving DNA which involves only single-stranded DNA. Furthermore, these structures revealed that TnpA recognizes both the ends of the mobile element and its integration target site through DNA-DNA interactions rather than using a site-specific DNA binding domain: target site recognition is accomplished by base pairing interactions between the target site and an internal segment of transposon DNA.

In bacteria, the movement of DNA by transposition is a crucial aspect of bacterial adaptation to new environments. For example, many bacterial transposons encode antibiotic resistance proteins in addition to those needed for their own mobility. We are interested in investigating the link between the emergence of antibiotic resistance and mobile genetic elements at the mechanistic level. Our current focus is on those transposons that move using a copy-out-paste-in mechanism, and we have recently solved by cryo-EM several structures corresponding to steps along this pathway. For the transposon we are studying, ISCth4, the structures revealed an unusual asymmetric dimeric transpososome in which an array of N-terminal domains from one monomer bind tightly to a single transposon end and the catalytic domains move to accommodate and direct the subsequent reactions.

The adeno-associated virus (AAV) is a small parvovirus that is able to integrate its genome site-specifically into human chromosome 19. This process appears unique to AAV, and is important because site-specific integration is a highly desirable property of vector systems used in gene therapy. To date, there are no site-specific systems available for clinical use. We believe that understanding how the AAV integration system works could provide ideas leading to the design of clinically useful systems. Our research focuses on the structural biochemistry of the AAV replication initiator protein, called Rep, that is not only needed for replication but also for site-specific integration. We have solved several structures of the site-specific DNA binding domain of Rep and its complexes with DNA. These have led us to propose a model for Rep assembly on DNA and the initiation of viral replication, which we continue to investigate in the context of both the full-length protein and the helicase domain.

A main focus in the laboratory is the mechanisms of eukaryotic DNA transposition. Transposition has been a fundamental shaping force during evolution. For instance, it has been established that over 50% of the human genome is transposon-derived. Recent efforts have been centered on the Hermes transposase from the insect species Musca domestica. Hermes is a member of one of the largest classes of eukaryotic transposons, the hAT transposons, which has representatives scattered among the genomes of insects, plants, and even humans. We have solved the three-dimensional structure of Hermes complexed with a segment of transposon end DNA which revealed an unexpected surprise: before DNA binding, Hermes is organized as a large ring of eight subunits, a very unusual arrangement for a DNA transposase. It appears that this octameric assembly is important so that Hermes can recognize its transposon ends which are several hundred basepairs long. More recently, we reported three X-ray structures of the Hermes transposase in complex with DNA that represent steps along the flanking hairpin-forming process. Our current studies with Hermes are focused on the question of why its transposition requires such long transposon ends.

PiggyBac, an active moth transposon, is a widely used transposon in genomic applications with a number of distinctive properties. Unlike other transposons, at the excision step it liberates itself by forming hairpins on its transposon ends while leaving behind precisely complementary single-stranded overhangs which can be repaired without leaving excision footprints. This property of "seamless excision" is very valuable in applications involving genome modifications to human cells. Also distinctive is that it integrates into new sites with a strict requirement for an TTAA target sequence. We have been probing the mechanistic basis of these excision and targeting properties, and recently solved the cryo-EM structures of the piggyBac transposase bound to its hairpin end and also at the step immediately following integration into a TTAA target. These structures provide the first insight into how the unusual transposition properties of piggyBac are structurally linked.

Another superfamily of eukaryotic DNA transposons that intrigues us are the Helitrons. Although no currently mobile Helitrons have been identified, they must once have been very active, as their remnants are widespread throughout the eukaryotic kingdom. Unlike other known eukaryotic DNA transposons, Helitron insertions in the host genome are not bordered by target site duplications, suggesting a replicative transposition mechanism that differs substantially from the cut-and-paste mode of transposition used by all other currently characterized eukaryotic systems. We have been investigating a reconstituted Helitron transposon, Helraiser, which is active in cells, and are building on in vivo assays and in vitro biochemical studies to work towards structural information.

Casposons have been proposed to be novel mobile elements that are the precursor of bacterial and archaeal adaptive immune systems. They contain genes encoding Cas1 homologs that we have shown are active DNA integrases, and we recently solved the X-ray crystal structure of a complex between a casposase, its target site, and its casposon ends. It seems that the casposase is remarkably eclectic in the types of substrates it will integrate but does so site-specifically. Our structural results led us to propose an evolutionary process in which an ancestral tetrameric casposase became the CRISPR-Cas integrase that is central to spacer integration.