Study of cellular responses to single-strand break

虽然单链断裂(Single-strand Break，SSB)频繁发生，但对单链断裂的细胞反应和修复还不是很清楚。为了解决这一问题，我们建立了表达粗糙脉孢菌UV损伤内切酶(UVDE)的哺乳动物细胞系，UVDE在UV诱导的环丁烷、嘧啶二聚体或6-4光产物中引入带有3‘-OH的SSB，立即引入5’-SSB，并启动另一种切除修复过程。表达UVDE的A组着色性干皮病细胞对紫外线的抗性几乎达到野生型水平。在这些细胞中，SSB在紫外线照射下产生，然后有效地修复。修复斑块大小约为7个核苷酸，修复合成减少到30%，提示δ/ε聚合酶参与了长斑块修复。紫外线照射后，细胞蛋白质立即聚合(ADP-核糖基)。在聚腺苷二磷酸核糖聚合酶(PARP)抑制剂3-氨基苯甲酰胺(3AB)的存在下，细胞的紫外线抗性降低。在XRCC1缺陷的中国仓鼠卵巢细胞系EM9中，UVDE的表达显著增加了宿主细胞对紫外线的敏感性，而3AB的加入几乎不会进一步增加细胞对紫外线的敏感性。因此，我们证明了XRCC1和PARP参与了SSB修复的相同途径。

Although single-strand breaks (SSBs) occur frequently, the cellular responses and repair of SSB are not well understood. To address this, we established mammalian cell lines expressing Neurospora crassa UV damage endonuclease (UVDE), which introduces a SSB with a 3'-OH, immediately 5' to UV-induced cycrobutane, pyrimidine dimers or 6-4 photoproducts and initiates an alternative excision repair process. Xeroderma pigmentosum group A cells expressing UVDE show UV resistance of almost the wild-type level. In these cells SSBs are produced upon UV irradiation and then efficiently repaired. The repair patch size is about 7 nucleotides and repair synthesis is decreased to 30% by aphidicoline, suggesting the involvement of a DNA polymerase δ/ε-dependent long-patch repair. Immediately after UV irradiation cellular proteins are poly(ADP-ribosyl)ated. The UV resistance of the cells is decreased in the presence of 3-aminobenzamide (3AB), an inhibitor of poly- (ADP-ribose) polymerase (PARP). Expression of UVDE in XRCC1-defective EM9, a Chinese hamster ovary cell line, greatly sensitizes the host cells to UV and addition of 3AB results in almost no further sensitization of the cells to UV. Thus, we show that XRCC1 and PARP are involved in the same pathway for the repair of SSBs.