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This study visualizes how G2L4 RT repairs DNA double-strand breaks using microhomology-mediated end joining (MMEJ). It reveals the enzyme

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Genome integrity relies on DNA double-strand break repair (DSBR).
  • Microhomology-mediated end joining (MMEJ) is an error-prone DSBR pathway with unclear mechanisms.
  • G2L4 RT, a bacterial group II intron-like reverse transcriptase, is implicated in MMEJ.

Purpose of the Study:

  • To elucidate the mechanistic details of G2L4 RT-mediated DSBR via MMEJ.
  • To visualize the dynamic process of MMEJ at the molecular level.

Main Methods:

  • High-speed atomic force microscopy (HS-AFM) was used for direct visualization.
  • Enzymatic assays were performed to observe DNA repair processes.

Main Results:

  • G2L4 RT dimers exhibit RT3a plug protrusion upon DNA binding.
  • G2L4 RT catalyzes MMEJ by stabilizing annealed microhomology and filling single-strand gaps.
  • Mn2+-stimulated terminal transferase activity generates branched DNA intermediates.
  • T4 DNA ligase seals nicks, stabilizing repaired products and preventing branching.

Conclusions:

  • G2L4 RT and T4 DNA ligase cooperatively shape MMEJ intermediates and outcomes.
  • Direct visualization reveals the step-by-step mechanism of G2L4 RT in MMEJ.
  • This study provides critical insights into an error-prone DNA repair pathway.