Retrotransposons hijack alt-EJ for DNA replication and eccDNA biogenesis

Retrotransposons are highly enriched in the animal genome 1 – 3 . The activation of retrotransposons can rewrite host DNA information and fundamentally impact host biology 1 – 3 . Although developmental activation of retrotransposons can offer benefits for the host, such as against virus infection, uncontrolled activation promotes disease or potentially drives ageing 1 – 5 . After activation, retrotransposons use their mRNA as templates to synthesize double-stranded DNA for making new insertions in the host genome 1 – 3 , 6 . Although the reverse transcriptase that they encode can synthesize the first-strand DNA 1 – 3 , 6 , how the second-strand DNA is generated remains largely unclear. Here we report that retrotransposons hijack the alternative end-joining (alt-EJ) DNA repair process of the host for a circularization step to synthesize their second-strand DNA. We used Nanopore sequencing to examine the fates of replicated retrotransposon DNA, and found that 10% of them achieve new insertions, whereas 90% exist as extrachromosomal circular DNA (eccDNA). Using eccDNA production as a readout, further genetic screens identified factors from alt-EJ as essential for retrotransposon replication. alt-EJ drives the second-strand synthesis of the long terminal repeat retrotransposon DNA through a circularization process and is therefore necessary for eccDNA production and new insertions. Together, our study reveals that alt-EJ is essential in driving the propagation of parasitic genomic retroelements. Our study uncovers a conserved function of this understudied DNA repair process, and provides a new perspective to understand—and potentially control—the retrotransposon life cycle.