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

The essential role of polymerase theta (Polθ)-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 Polθ 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 Polθ 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.