Retrotransposons are mobile genetic elements in the human genome that are recognized as drivers of genome expansion and evolution. The Long Interspersed Element-1 (LINE-1) retrotransposon has generated over one-third of the human genome and serves as an active source of genetic diversity and human disease. Yet, how LINE-1 mobilizes within the human genome remains poorly understood. I will discuss our efforts to biochemically reconstitute the mobility mechanism by the human LINE-1 encoded enzyme with purified components. Our reconstitutions demonstrate how the LINE-1 enzyme nicks the target DNA to prime reverse transcription of the LINE-1 or SINE RNAs in vitro.

Using cryo-electron microscopy, we have obtained structures of retrotransposition intermediates with the LINE-1 enzyme engaging its native RNAs, e.g. the SINE RNAs, and target DNA to prime reverse transcription. We visualize extensive interactions with the single-stranded RNA and RNA secondary structures by five distinct domains of the LINE-1 enzyme, including sequence-specific contacts. Most surprisingly, we demonstrate an unexpected target-site requirement for DNA cleavage and reverse transcription where the enzyme recognizes an upstream single-stranded DNA to position adjacent DNA duplex in the endonuclease active site for nicking, generating a staggered DNA break with a single nick. These findings demonstrate that LINE-1's mobility is coupled to the DNA replication within human cells.

In summary, our work provides key insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.