What is it about?

Through this study, we determined the 3D-structures of group II intron–like reverse transcriptase‑4 (G2L4 RT), an enzyme derived from a group II intron reverse transcriptase that has been repurposed by bacteria for DNA repair. This work connects directly to the DNA repair function of G2L4 RT reported previously (Park et al., 2022), adding significance by the structural basis mechanism behind this function. In its inactive state, G2L4 RT forms a homodimer, with its catalytic site blocked by a short helix‑turn‑helix structure named the RT3a plug. This RT3a plug represents a new conformation that was not predicted by AlphaFold3. Upon encountering a DNA substrate, G2L4 RT remains a dimer, an extended N-terminal extension RT0 loop structure that grips DNA ends, stabilizing them for microhomology‑mediated end joining (MMEJ) DNA repair. When the RT0 loop or dimer features were disrupted by mutagenesis, the enzyme almost completely lost its ability to repair DNA breaks, confirming the functional predictions made by the structure. Unlike mobile group II intron RTs, which exist as monomeric RNP complexes, G2L4 RT evolved amino acid substitutions in its catalytic site, co‑variations at adjacent residues, and contributions from the thumb domain to the dimer interface—changes that collectively converted an intron‑mobilizing enzyme into one specialized for DNA repair. These findings provide structural insight into how a retroelement‑derived enzyme was reshaped by evolution for a new role in maintaining genome stability. Reference Park SK, Mohr G, Yao J, Russell R, Lambowitz AM. Group II intron-like reverse transcriptases function in double-strand break repair. Cell. 2022 Sep 29;185(20):3671-3688.e23. doi: 10.1016/j.cell.2022.08.014. Epub 2022 Sep 15. PMID: 36113466; PMCID: PMC9530004.

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Why is it important?

Because DNA damage drives genome instability and cell apoptosis, understanding how cells repair such damage is crucial. This study identified a previously unknown DNA repair enzyme and explained the structural basis for its functional adaptation. Importantly, these findings offer insight into how other RTs, including human LINE‑1 RT, which is active in many cancers and aging cells, might similarly interact with DNA breaks and influence DNA repair pathways. This discovery could also enable the design of engineered enzymes to guide more precise CRISPR editing or to slow harmful DNA repair processes in diseases like cancer. More broadly, this work demonstrates that RT functions extend far beyond those of viruses and retroelements, suggesting that many additional “repurposed enzymes” may exist in nature. By combining structural biology, evolutionary analysis, and biochemical approaches, this study opens new directions for both basic research and biotechnological applications.

Perspectives

Although retroviral reverse trancriptases (RTs) are the most widely known, RTs are ancient enzymes that evolved from an RNA-dependent-DNA polymerase, likely during the transition from an RNA to DNA world. They remain prevalent in bacteria where they are associated retroelements called mobile group II introns, which were evolutionary ancestors of eukaryotic spliceosomal introns and key components of the eukaryotic splicing apparatus, as well as retroviral and other eukaryotic RTs. Bacteria also harbor numerous chromosomally encoded domesticated RTs that evolved from group II intron RTs to perform different cellular functions, including phage defense by multiple mechanisms, host-phage tropism switching that impacts phage infection, and RNA spacer acquisition in CRISPR-Cas systems. We recently identified a family of domesticated bacterial group II intron-like RTs, denoted G2L4 RTs, that evolved to function in cellular double-strand break repair and also found this to be a basal activity of group II intron-encoded RTs. Structural analysis revealed a series of adaptations of a G2L4 RT that optimized this cellular function, including a plug-like structure that stably blocks the RT active site until encountering a physiological substate; modifications of the RT active site and other regions that favor strand-annealing over processive DNA synthesis; and a dimerization interface that localizes two G2L4 RT monomers to the same double-strand break site, positioned to carry out different functions in double-strand DNA break repair by microhomology-mediated end-joining . These findings revealed how G2L4 RTs evolved to function optimally in DNA repair, raised the possibility that human LINE-1 and other related RTs have a similar function, and suggested ways of optimizing RTs for genome engineering applications.

Dr. Seung Kuk Park
Stanford University

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This page is a summary of: Structural basis for the evolution of a domesticated group II intron–like reverse transcriptase to function in host cell DNA repair, Proceedings of the National Academy of Sciences, July 2025, Proceedings of the National Academy of Sciences,
DOI: 10.1073/pnas.2504208122.
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