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PreprintCC BY-NC-NDbioRxiv

ssDNA hand-off from RPA to Polθ drives microhomology-mediated end-joining

  • Martha J. Longley
  • Hyunjee Lee
  • Owen Eby
  • Bhasker Dharavath
  • Yufan Wu
  • Abhilash Jayaraj
  • Felix Steinruecke
  • Haribabu Arthanari
  • Ralph Scully
  • Lucas Farnung
  • Joseph J. Loparo
  • Johannes C. Walter
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Abstract

DNA polymerase theta-mediated end joining (TMEJ) is the primary mechanism of double strand break repair in mitosis and becomes essential in BRCA-deficient cells. RPA, the ssDNA-binding protein that coats resected DNA ends, has long been thought to inhibit this pathway by blocking the initial microhomology search in TMEJ. Here we find instead that RPA plays an essential activating role in TMEJ. Structural, biochemical, and genetic analyses support a model in which Pol{theta} binds directly to RPA at resected DNA ends and then competes with RPA for ssDNA. This releases ssDNA into the Pol{theta} helicase domain. We propose that threading two DNA ends through the Pol{theta} dimer in this manner initiates the search for microhomology. Together, our findings establish a detailed mechanism of how proteins can harness RPA to access ssDNA for critical genome maintenance transactions.

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Cite

BibTeXRIS
BibTeX
@misc{longley2026ssdna,
  title = {{ssDNA hand-off from RPA to Polθ drives microhomology-mediated end-joining}},
  author = {Martha J. Longley and Hyunjee Lee and Owen Eby and Bhasker Dharavath and Yufan Wu and Abhilash Jayaraj and Felix Steinruecke and Haribabu Arthanari and Ralph Scully and Lucas Farnung and Joseph J. Loparo and Johannes C. Walter},
  howpublished = {bioRxiv},
  year = {2026},
  month = oct,
  doi = {10.64898/2026.10.26.762034},
  url = {https://www.biorxiv.org/content/10.64898/2026.10.26.762034v1},
  note = {CC BY-NC-ND},
}
RIS
TY  - UNPB
TI  - ssDNA hand-off from RPA to Polθ drives microhomology-mediated end-joining
AU  - Martha J. Longley
AU  - Hyunjee Lee
AU  - Owen Eby
AU  - Bhasker Dharavath
AU  - Yufan Wu
AU  - Abhilash Jayaraj
AU  - Felix Steinruecke
AU  - Haribabu Arthanari
AU  - Ralph Scully
AU  - Lucas Farnung
AU  - Joseph J. Loparo
AU  - Johannes C. Walter
PY  - 2026
DA  - 2026/10/26
PB  - bioRxiv
DO  - 10.64898/2026.10.26.762034
UR  - https://www.biorxiv.org/content/10.64898/2026.10.26.762034v1
AB  - DNA polymerase theta-mediated end joining (TMEJ) is the primary mechanism of double strand break repair in mitosis and becomes essential in BRCA-deficient cells. RPA, the ssDNA-binding protein that coats resected DNA ends, has long been thought to inhibit this pathway by blocking the initial microhomology search in TMEJ. Here we find instead that RPA plays an essential activating role in TMEJ. Structural, biochemical, and genetic analyses support a model in which Pol{theta} binds directly to RPA at resected DNA ends and then competes with RPA for ssDNA. This releases ssDNA into the Pol{theta} helicase domain. We propose that threading two DNA ends through the Pol{theta} dimer in this manner initiates the search for microhomology. Together, our findings establish a detailed mechanism of how proteins can harness RPA to access ssDNA for critical genome maintenance transactions.
SN  - 2692-8205
ER  -