Replication fork stalling can provoke fork reversal to form a four-way DNA junction. This remodelling of the replication fork can facilitate repair, aid bypass of DNA lesions, and enable replication restart, but may also pose a risk of over-replication during fork convergence. We show that replication fork stalling at a site-specific barrier in fission yeast can induce gene duplication-deletion rearrangements that are independent of replication restart-associated template switching and Rad51-dependent multi-invasion. Instead, they resemble targeted gene replacements (TGRs), requiring the DNA annealing activity of Rad52, the 3’-flap nuclease Rad16-Swi10, and mismatch repair protein Msh2. We propose that excess DNA, generated during the merging of a canonical fork with a reversed fork, can be liberated by a nuclease and integrated at an ectopic site via a TGR-like mechanism. This highlights how over-replication at replication termination sites can threaten genome stability in eukaryotes.

复制叉停滞会引发复制叉逆转，形成四向 DNA 连接。复制叉的这种重塑可以促进修复，帮助绕过 DNA 损伤，并使复制重新启动，但也可能在叉收敛期间带来过度复制的风险。我们发现，在裂殖酵母中，复制叉在位点特异性屏障处停滞可以诱导基因重复删除重排，该重排独立于复制重启相关的模板切换和依赖于 Rad51 的多重入侵。相反，它们类似于靶向基因替换 (TGR)，需要 Rad52、3'-瓣核酸酶 Rad16-Swi10 和错配修复蛋白 Msh2 的 DNA 退火活性。我们提出，在正则叉与反向叉合并过程中产生的多余 DNA 可以被核酸酶释放，并通过类似 TGR 的机制整合到异位位点。这凸显了复制终止位点的过度复制如何威胁真核生物基因组的稳定性。