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 have recently solved its structure, alone and in complex with a short DNA hairpin sequence that is located near each transposon end. The binding of TnpA to these subterminal hairpins is how TnpA finds and pairs the transposon ends before ISHp608 excision. We have now been able to crystallize TnpA with several other DNA molecules representing subsequent steps along the transposition pathway.

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 the full-length protein.

We are also studying 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. We have recently solved the high resolution three-dimensional structure of 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. The structure revealed an unexpected surprise: Before DNA binding, Hermes is organized as a hexamer, a very unusual arrangement for a DNA transposase. We are currently attempting to understand the functional implications of the hexameric assembly and to determine the structure of Hermes bound to its DNA substrates.

We also study how 14-3-3 proteins, a family of highly conserved homo- and heterodimeric proteins, conformationally modulate their binding partners. 14-3-3 proteins are key regulatory elements in a wide variety of biological processes. Their regulatory effect is mediated by their ability to bind other proteins in a phosphorylation-dependent manner. One such binding partner is serotonin N-acetyltransferase, the key enzyme involved in the biosynthesis of the circadian neurohormone, melatonin. We have shown that during catalysis, the enzyme undergoes a conformational change, the nature of which explains its kinetic properties. When the enzyme is phosphorylated, it binds tightly to 14-3-3, and in the resulting complex, the enzyme's conformation is stabilized in a configuration that increases its binding affinity for its substrates. This - in turn - increases the enzyme's activity.

Last but not least, we have a long-standing interest in how the HIV virus integrates its DNA into the human genome. Integration of viral DNA is the irreversible step of retroviral infection during which the virus splices its DNA into that of its host. We have been investigating the structures of host cell proteins that assist or direct viral integration. We recently determined the structures of (i) BAF, the Barrier-to-Autointegration Factor, bound to DNA, and (ii) the dimerization domain of LAP2alpha. Both of these proteins have been identified as components of retroviral preintegration complexes, and we hope that their structures will help us to better understand how they affect integration.

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