PROJECT SUMMARY A key step in the assembly of the large double-stranded DNA (dsDNA) viruses is packaging of a genome into a pre-assembled procapsid by an ATP-driven motor complex. In the herpesviruses and many bacteriophages, packaging is catalyzed by a terminase enzyme that utilizes a concatemeric genome substrate. To accomplish this, terminase enzymes assemble into distinct initiation, motor and termination complexes to processively excise an individual genome from the concatemer, and concomitantly package it into the capsid. This requires that the enzymes cycle between stable nuclease and dynamic motor intermediates. While our understanding of packaging initiation and motor translocation is extensive, termination of genome packaging remains ill-studied and poorly characterized in all viruses, primarily because defined experimental systems have not been developed. Phage is an exception wherein rigorous biochemical assays allow molecular dissection of the entire assembly pathway. This multi-PI application proposes to use phage to interrogate termination, the final and most poorly characterized step in the packaging pathway. Two fundamental questions central to genome packaging are addressed; (i) how does the translocating motor recognize the genome end while also sensing that a sufficient length of DNA has been packaged, transition to a site-specifically bound nuclease complex, and (ii) how do “finishing proteins” promote end maturation and terminase ejection from the nucleocapsid without loss of the tightly packaged DNA. We describe highly integrated and synergistic biochemical, biophysical, single-molecule and structural approaches to characterize this conserved and essential, yet largely unstudied step in virus assembly. Given that this process is strongly conserved in all of the dsDNA viruses, both prokaryotic and eukaryotic, and the commonality of initiation-translocation-termination pathways in biology, the results will have broad implications in virology and cell biology.