TONSL suppresses polymerase theta-dependent tandem duplications through chromatin-guided repair

Tandem duplications (TDs) are a common form of genomic rearrangements with both adaptive and pathogenic consequences. While prevalent in genomically unstable cancer genomes, TDs are rarely detected in normal tissues, suggesting the existence of robust protective mechanisms. Here, we identify the histone chaperone TONSL/TONSOKU (tnsl-1 in C. elegans) as a critical suppressor of TD formation. Loss of tnsl-1 results in the accumulation of TDs in two distinct size classes (~25 kb and ~300 kb), arising from different developmental contexts: small TDs emerge in rapidly dividing embryonic cells, whereas large TDs form in slower-dividing germline progenitors. Both classes depend on polymerase theta-mediated end joining (TMEJ), implicating DNA double-strand breaks in their genesis. Inhibition of break-induced replication (BIR) via Pif1 helicase loss reduces TD size, revealing a role for BIR in TD expansion. Remarkably, TONSL-deficient Arabidopsis thaliana exhibit an identical TD signature, highlighting the evolutionary conservation of this genome surveillance mechanism. These findings position TONSL as a cross-kingdom guardian of genome integrity through suppression of TD formation. Tandem duplications are a common class of structural variation in evolving genomes but are rare in healthy cells, and the DNA repair mechanisms that suppress their formation remain poorly defined. Here, the authors identify TONSL as a conserved factor that suppresses tandem duplications across species.