Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

染色体脆弱位点与促进基因组不稳定性有关，这会导致癌症和神经系统疾病。然而，染色体脆性的原因和机制仍然是推测性的。在这里，我们确定了大肠杆菌中的三个自发脆弱位点基因组，并以高分辨率定义它们的 DNA 损伤和修复中间体。我们发现所有三个位点都位于复制终止区域，显示出反复出现的四向 DNA 或 Holliday 连接 (HJ) 和反复出现的 DNA 断裂。同源定向双链断裂修复在所有这些位点产生复发的 HJ；然而，DNA断裂的不同机制是牵连的：复制叉在自然复制屏障处塌陷，并且出乎意料的是，在细胞分裂时未分离的姐妹染色体频繁剪切。我们提出，这两种机制可能普遍存在，包括人类，并且可能构成体细胞嵌合、癌症和其他基因组不稳定疾病的一些最早事件。