Lesions initiating spontaneous mitotic crossover are minimally subject to non-homologous end joining.

Homologous recombination (HR) between sister chromatids is the dominant outcome of replication-associated DNA repair, yet the lesions that initiate spontaneous mitotic crossovers remain poorly defined. Most mechanistic work on HR pathway choice uses enzymatically induced two-ended double-strand breaks (DSBs), where non-homologous end joining (NHEJ) is a major competing pathway. Whether NHEJ also competes for spontaneous, replication-associated lesions has not been directly tested. Here we use sci-L3-Strand-seq, a single-cell replication-template strand-specific sequencing method that maps sister chromatid exchange (SCE) genome-wide, to profile heterozygous and homozygous knockouts of NHEJ factors (LIG4, XRCC4) and the single-strand break (SSB) repair scaffold XRCC1 in HAP1 and BJ cell lines. NHEJ disruption produced only a modest (30%) increase in SCE, whereas XRCC1 loss caused a pronounced, 5-fold elevation. The dense, widespread SCE pattern in XRCC1-deficient cells is consistent with unrepaired SSBs being converted at replication forks into one-ended DSBs that lack a second end for ligation and therefore cannot engage NHEJ. In parallel, structural variation (SV) mapping in the same single cells revealed a strongly non-random landscape dominated by recurrent chromosome losses and clonal expansion, indicating selective pressure and stepwise genome evolution. SCE frequency did not correlate with SV burden or SV-defined subclones, demonstrating that error-free recombination and mutational rearrangement represent separable axes of genome maintenance. Recovery of reciprocal daughter-cell pairs with matching SCE breakpoints directly confirms that these events arose by inter-sister exchange in the preceding division. Together, these results show that spontaneous mitotic crossovers are driven by lesions largely incompatible with NHEJ and instead engage HR through replication-coupled SSB-to-DSB conversion, and that elevated error-free recombination is decoupled from the mutational SV landscape.