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<p>Homologous recombination (HR) repairs DNA double-strand breaks and stabilizes stressed replication forks, and HR deficiency promotes genome instability and cancer. HR requires assembly of RAD51 nucleoprotein filaments on single-stranded DNA (ssDNA), a process regulated by the human RAD51 paralogs RAD51C, XRCC3, RAD51D and XRCC2. Here, using cryo-electron microscopy, we find that the RAD51–XRCC3–RAD51C complex (RAD51–X3C) assembles into an octamer in which XRCC3 engages the RAD51 DNA-binding surface and RAD51 subunits adopt a misaligned configuration incompatible with filament formation. These features define an autoinhibited RAD51–X3C state that limits nonproductive RAD51 binding to double-stranded DNA or RNA–DNA hybrids while preserving RAD51 availability for ssDNA-dependent strand exchange. We further show that the RAD51D–XRCC2 paralog complex remodels RAD51–X3C into a pentameric RAD51–X3CDX2 assembly by engaging the exposed RAD51C surface and disrupting contacts that stabilize the octamer. This remodeling exposes the RAD51 DNA-binding interface, enhances RAD51–ssDNA filament assembly, and promotes strand exchange on RPA-coated ssDNA, and yields a filament-compatible paralog assembly that integrates into ssDNA-bound RAD51 filaments. Together, these findings establish paralog exchange as a mechanism that converts an autoinhibited RAD51–X3C octamer into an activated RAD51–X3CDX2 pentamer to regulate RAD51 filament formation during HR and replication fork preservation.</p>

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Structural insight into how RAD51 paralog exchange regulates RAD51 filament formation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月22日

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  • Yashpal Rawal,
  • Youngho Kwon,
  • Lijia Jia,
  • Eliza A. Ruben,
  • Jae-Hoon Ji,
  • Lijuan Guo,
  • Caleb M. Stratton,
  • Digant Nayak,
  • Miriam Tovar,
  • Qingming Fang,
  • Mohd Azrin Jamalruddin,
  • Shuo Zhou,
  • Sahiti Kuppa,
  • Shahrez Syed,
  • Angela M. Jasper,
  • Jeffrey N. Katz,
  • Cody M. Rogers,
  • Hardeep Kaur,
  • Lorena Samentar,
  • Weixing Zhao,
  • Eloise Dray,
  • Fang Zhang,
  • Svetla Stoilova-McPhie,
  • Alexander B. Taylor,
  • Sandeep Burma,
  • Manjeet K. Rao,
  • David S. Libich,
  • Robert Hromas,
  • Alexander V. Mazin,
  • Maria Jasin,
  • Daohong Zhou,
  • Kara A. Bernstein,
  • Eric C. Greene,
  • Elizabeth V. Wasmuth,
  • Patrick Sung,
  • Shaun K. Olsen

摘要

Homologous recombination (HR) repairs DNA double-strand breaks and stabilizes stressed replication forks, and HR deficiency promotes genome instability and cancer. HR requires assembly of RAD51 nucleoprotein filaments on single-stranded DNA (ssDNA), a process regulated by the human RAD51 paralogs RAD51C, XRCC3, RAD51D and XRCC2. Here, using cryo-electron microscopy, we find that the RAD51–XRCC3–RAD51C complex (RAD51–X3C) assembles into an octamer in which XRCC3 engages the RAD51 DNA-binding surface and RAD51 subunits adopt a misaligned configuration incompatible with filament formation. These features define an autoinhibited RAD51–X3C state that limits nonproductive RAD51 binding to double-stranded DNA or RNA–DNA hybrids while preserving RAD51 availability for ssDNA-dependent strand exchange. We further show that the RAD51D–XRCC2 paralog complex remodels RAD51–X3C into a pentameric RAD51–X3CDX2 assembly by engaging the exposed RAD51C surface and disrupting contacts that stabilize the octamer. This remodeling exposes the RAD51 DNA-binding interface, enhances RAD51–ssDNA filament assembly, and promotes strand exchange on RPA-coated ssDNA, and yields a filament-compatible paralog assembly that integrates into ssDNA-bound RAD51 filaments. Together, these findings establish paralog exchange as a mechanism that converts an autoinhibited RAD51–X3C octamer into an activated RAD51–X3CDX2 pentamer to regulate RAD51 filament formation during HR and replication fork preservation.