DNA lesion bypass facilitates DNA synthesis across bulky DNA lesions, playing a critical role in DNA damage tolerance and cell survival after DNA damage. Assessing lesion bypass efficiency in the cell is important to better understanding of the mechanism of carcinogenesis and chemoresistance. Here we developed a chromatin immunoprecipitation (ChIP)-based method to measure lesion bypass activity across cisplatin-induced intrastrand crosslinks in cancer cells. DNA lesion bypass enables the replication to continue in the presence of replication blocks. Thus, the successful lesion bypass should result in the coexistence of DNA lesions and the newly synthesized DNA fragment opposite to this lesion. Using ChIP, we precipitated the cisplatin-induced intrastrand crosslinks, and quantitated the precipitated newly synthesized DNA that was labeled with BrdU. We validated this method on ovarian cancer cells with inhibited TLS activity. We then applied this method to show that ovarian cancer stem cells exhibit high lesion bypass activity relative to bulk cancer cells from the same cell line. In conclusion, this novel ChIP-based lesion bypass assay can detect the extent to which cisplatin-induced DNA lesions are bypassed in live cells. Our study may be applied more broadly to the study of other DNA lesions, as specific antibodies to these specific lesions are available.

DNA 损伤旁路促进 DNA 合成跨越庞大的 DNA 损伤，在 DNA 损伤耐受性和 DNA 损伤后的细胞存活中发挥关键作用。评估细胞中的病变旁路效率对于更好地理解癌变和化学抗性的机制很重要。在这里，我们开发了一种基于染色质免疫沉淀 (ChIP) 的方法来测量癌细胞中顺铂诱导的链内交联的病变绕过活性。DNA 损伤旁路使复制能够在存在复制块的情况下继续进行。因此，成功的损伤绕过应该导致 DNA 损伤和与该损伤相反的新合成 DNA 片段共存。使用 ChIP，我们沉淀了顺铂诱导的链内交联，并对沉淀的新合成的用 BrdU 标记的 DNA 进行了定量。我们在具有抑制 TLS 活性的卵巢癌细胞上验证了这种方法。然后我们应用这种方法来证明卵巢癌干细胞相对于来自同一细胞系的大量癌细胞表现出高病变旁路活性。总之，这种基于 ChIP 的新型损伤绕过检测可以检测活细胞中顺铂诱导的 DNA 损伤被绕过的程度。我们的研究可能更广泛地应用于其他 DNA 损伤的研究，因为可以使用针对这些特定损伤的特异性抗体。这种基于 ChIP 的新型损伤旁路检测可以检测顺铂诱导的 DNA 损伤在活细胞中被旁路的程度。我们的研究可能更广泛地应用于其他 DNA 损伤的研究，因为可以使用针对这些特定损伤的特异性抗体。这种基于 ChIP 的新型损伤旁路检测可以检测顺铂诱导的 DNA 损伤在活细胞中被旁路的程度。我们的研究可能更广泛地应用于其他 DNA 损伤的研究，因为可以使用针对这些特定损伤的特异性抗体。