A direct RPA-Polθ interaction licenses theta-mediated end joining
- 1Leiden University Medical Center
Abstract
DNA double-strand break repair often involves end resection, yet how resected ends are directed toward distinct repair pathways remains poorly understood. Here, we identify replication protein A (RPA) as an unexpected essential factor for cellular polymerase theta-mediated end joining (TMEJ). Using MUSIC-MAP, a newly developed platform linking endogenous sequence variants to DNA repair outcomes, we map a discrete functional interface between RPA2 and polymerase theta (Pol{theta}). Disrupting this interface in RPA2 abolishes TMEJ without impairing cell viability or homologous recombination. Mutations in the complementary Pol{theta} surface likewise impair TMEJ, whereas reciprocal charge-reversal mutations in RPA2 and Pol{theta} restore TMEJ, providing genetic evidence for a direct physical interaction. Structural modeling suggests that the Pol{theta}-binding surface becomes accessible at RPA-coated ssDNA termini, positioning RPA as a docking platform for Pol{theta} engagement. These findings reveal how RPA, previously considered a barrier to TMEJ, instead functions as a molecular platform that translates the organization of ssDNA into selective enzyme engagement, enabling mutagenic repair without compromising homologous recombination.
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BibTeX
@misc{barazas2026direct,
title = {{A direct RPA-Polθ interaction licenses theta-mediated end joining}},
author = {M. Barazas and C. I. Bos and H. Kool and J. Schimmel and R. van Schendel and M. S. Luijsterburg and M. Tijsterman},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.25.761560},
url = {https://www.biorxiv.org/content/10.64898/2026.10.25.761560v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A direct RPA-Polθ interaction licenses theta-mediated end joining
AU - M. Barazas
AU - C. I. Bos
AU - H. Kool
AU - J. Schimmel
AU - R. van Schendel
AU - M. S. Luijsterburg
AU - M. Tijsterman
PY - 2026
DA - 2026/10/25
PB - bioRxiv
DO - 10.64898/2026.10.25.761560
UR - https://www.biorxiv.org/content/10.64898/2026.10.25.761560v1
AB - DNA double-strand break repair often involves end resection, yet how resected ends are directed toward distinct repair pathways remains poorly understood. Here, we identify replication protein A (RPA) as an unexpected essential factor for cellular polymerase theta-mediated end joining (TMEJ). Using MUSIC-MAP, a newly developed platform linking endogenous sequence variants to DNA repair outcomes, we map a discrete functional interface between RPA2 and polymerase theta (Pol{theta}). Disrupting this interface in RPA2 abolishes TMEJ without impairing cell viability or homologous recombination. Mutations in the complementary Pol{theta} surface likewise impair TMEJ, whereas reciprocal charge-reversal mutations in RPA2 and Pol{theta} restore TMEJ, providing genetic evidence for a direct physical interaction. Structural modeling suggests that the Pol{theta}-binding surface becomes accessible at RPA-coated ssDNA termini, positioning RPA as a docking platform for Pol{theta} engagement. These findings reveal how RPA, previously considered a barrier to TMEJ, instead functions as a molecular platform that translates the organization of ssDNA into selective enzyme engagement, enabling mutagenic repair without compromising homologous recombination.
ER -