Hierarchical Coordination of Polymerase Theta and RAD51 Resolves Clustered Replication Fork Collapse

Smith, C.; Simpson, D. A.; Guerra, P.; Feng, W.; Ellington, S. W.; Lu, D.; Fijen, C.; Boylston, N. M.; Chriuevella, K. K.; El-Shiekh, I.; Lee, R.; Wozny, A.-S.; Pregnall, A. M.; Eckenroth, B. E.; El-Sagheer, A. H.; Brown, T.; Doublie, S.; Ramsden, D. A.; Rothenberg, E.; Nik-Zainal, S.; Gupta, G. P.

doi:10.1101/2025.07.02.662846

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bioRxiv · 10.1101/2025.07.02.662846

Hierarchical Coordination of Polymerase Theta and RAD51 Resolves Clustered Replication Fork Collapse

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Abstract

The essential role of polymerase theta (Pol{theta})-mediated end joining (TMEJ), an alternative double strand break repair pathway, has been primarily studied in homologous recombination (HR)-deficient contexts(1, 2). Here, we uncover an indispensable role for TMEJ in HR-proficient mammalian cells during the repair of interstrand crosslinks (ICLs). We show that Pol{theta} is recruited downstream of canonical ICL repair steps--including ICL unhooking, RAD51 loading, and RAD51 ubiquitylation--and localizes to sites of unresolved HR through interactions with ubiquitylated RAD51 filaments. Using genomic scar profiling and targeted ICL repair assays, we find that TMEJ resolves a minor subset of lesions that are not amenable to HR repair, such as clustered ICLs that can induce two-ended replication fork collapse. These findings reveal a RAD51 ubiquitylation-dependent mechanism for Pol{theta} recruitment and establish TMEJ as a hierarchically deployed DNA repair pathway that safeguards genome stability when HR is insufficient to resolve replication-associated DNA damage. Short SummaryPol{theta} is recruited via RAD51 ubiquitylation to resolve clustered ICLs that generate HR-refractory replication fork collapse.

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Smith, C., Simpson, D. A., Guerra, P., Feng, W., Ellington, S. W., Lu, D., Fijen, C., Boylston, N. M., Chriuevella, K. K., El-Shiekh, I., Lee, R., Wozny, A.-S., Pregnall, A. M., Eckenroth, B. E., El-Sagheer, A. H., Brown, T., Doublie, S., Ramsden, D. A., Rothenberg, E., Nik-Zainal, S., Gupta, G. P.. 2025-07-03. Hierarchical Coordination of Polymerase Theta and RAD51 Resolves Clustered Replication Fork Collapse. https://doi.org/10.1101/2025.07.02.662846

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