Long-term XPC silencing reduces DNA double-strand break repair
Long-term XPC silencing reduces DNA double-strand break repair
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DOI：
10.1158/0008-5472.can-06-3371
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发表时间：
2007-03-15
期刊：
影响因子：
11.2
通讯作者：
Biard, Denis S. F.
中科院分区：
文献类型：
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作者：
Despras, Emmanuelle;Pfeiffer, Petra;Biard, Denis S. F.
为了研究不同 DNA 修复途径之间的关系，我们建立了一组克隆，其中使用长期 RNA 干扰在 HeLa 细胞系中沉默了一个特定的 DNA 修复基因。我们在这里关注参与核苷酸切除修复（XPA 和 XPC）或非同源末端连接（NHEJ；DNA-PKcs 和 XRCC4）的基因。正如预期的那样，XPA(KD)（敲低）和 XPCKD 细胞对 UVC 高度敏感。 DNA-PKcs(KD) 和 XRCC4(KD) 细胞对各种双链断裂 (DSB) 诱导剂的敏感性增加，体外 NHEJ 活性降低 70% 至 80%。 XPC 基因表达的长期沉默导致对依托泊苷的敏感性增加，依托泊苷是一种拓扑异构酶 II 抑制剂，可通过 DNA 复制叉的进展产生 DSB。 XPCKD 细胞还表现出对急性伽马射线照射的不耐受。我们发现 XPCKD 细胞表现出 NHEJ 产物谱的改变，分子内连接产物的水平降低。此外，在 XPCKD 和 DNA-PKcs(KD) 细胞中，XRCC4 和连接酶 IV 蛋白在较低剂量的 DSB 诱导剂下被动员到受损的核结构上。在 XPC 熟练的细胞中，XPC 蛋白在 DSB 诱导后从核结构中释放出来。相比之下，XPA 基因表达的沉默对 DSB 或 NHEJ 的敏感性没有任何影响。我们的结果表明，XPC 缺陷，当然与其他遗传缺陷相结合，可能会损害 DSB 修复。
To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPA(KD) (knock down) and XPCKD cells were highly sensitive to UVC. DNA-PKcs(KD) and XRCC4(KD) cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPCKD cells also showed intolerance toward acute gamma-ray irradiation. We showed that XPCKD cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPCKD and DNA-PKcs(KD) cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair.