Distinct repair outcomes from single and convergent replication fork collapse

Replication fork collapse at single-strand DNA breaks threatens genome stability but how such forks are repaired and resolved has remained unclear. Here we replicate site-specific nicks with single or converging replication forks in Xenopus laevis egg extracts. Collapse of a single fork generates a single-ended double-strand break (DSB) that undergoes homologous recombination to yield stable D-loops and end-to-end fusions, yet does not restart DNA synthesis. Single collapsed forks can also undergo extensive nucleolytic degradation, appearing to disassemble the sister fork through ‘secondary collapse’ events that resolve single-ended DSBs without engaging DSB repair. In contrast, semisynchronous convergent collapse generates a double-ended DSB that is primarily repaired through annealing-dependent DSB repair, completing DNA synthesis but generating precise deletions and templated insertions. These error-prone products are not detected following single-fork collapse. Our findings demonstrate that single and semisynchronous convergent collapsed forks elicit distinct repair outcomes.