Serine integrases carry out recombination between short, specific DNA sequences commonly referred to as attP and attB to integrate a viral genome into the chromosome of a host. The reaction does not require accessory proteins, additional DNA elements, or other cofactors and is effectively irreversible in the absence of a viral recombination directionality factor (RDF) protein. These features have led to the emergence of serine integrases as important tools in genome engineering and synthetic biology applications. As bacteriophage integrases, the serine integrases have a strong influence over microbial communities, including the human gut microbiota, where the resident phage population is thought to influence human health. Serine integrases are also responsible for the horizontal transfer of antibiotic resistance genes within bacterial communities. Despite their importance in human health and their widespread use in biomedical research, our mechanistic understanding of how serine integrases function is still primitive. To address the largest gaps in our understanding of serine integrase structure and function, we will determine the first structure of an integrase- attachment site structure, establish the structural basis for RDF function, and test the hypothesis that serine integrase efficiency in mammalian cells is limited by the strength of attP and attB site association. Our experimental approaches include a novel kinetic analysis of the integration reaction, structure determination of reaction intermediates using X-ray diffraction, analytical ultracentrifugation, novel integration and excision assays, and integration into chromosomal sites in human cells. Our functional model for serine integrase regulation of site-specificity, directionality, and site-association using interactions between coiled-coil domains is unprecedented among nucleic acid enzymes. These studies will provide an important framework for engineering improved function in serine integrase applications and may lead directly to improved integrases for targeted integration in mammalian cells.