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bioRxiv · 10.64898/2026.03.13.711756

Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining

Zhang, P.·Guo, M.·Zhang, Y. J.·Lambowitz, A. M.·Lin, Y.-C.
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Abstract

Double-strand break repair (DSBR) is essential for genome integrity, yet mechanistic details of error-prone microhomology-mediated end joining (MMEJ) remain unclear. A bacterial group II intron-like reverse transcriptase, G2L4 RT, has been implicated in MMEJ, but how it executes DSBR is unknown. Using high-speed atomic force microscopy (HS-AFM), we directly visualize G2L4 RT-mediated DSBR via MMEJ. We observe that G2L4 RT dimers exhibit RT3a plug protrusion upon DNA engagement and catalyze MMEJ by binding and stabilizing a 4-bp annealed microhomology and filling adjacent single-strand gaps with dNTPs. We also observe Mn2+-stimulated terminal transferase activity that generates elongated and branched DNA intermediates prior to ligation. With T4 DNA ligase, we visualize binding near nick sites and real-time nick sealing, which stabilizes the repaired products and suppresses off-pathway branching. These results reveal how G2L4 RT and ligase activities shape MMEJ intermediates and outcomes.

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Zhang, P., Guo, M., Zhang, Y. J., Lambowitz, A. M., Lin, Y.-C.. 2026-03-17. Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining. https://doi.org/10.64898/2026.03.13.711756

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