DNA damage repair genes controlling human papillomavirus (HPV) episome levels under conditions of stability and extreme instability.
DNA damage repair genes controlling human papillomavirus (HPV) episome levels under conditions of stability and extreme instability.
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DOI：
10.1371/journal.pone.0075406
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Fisher C
中科院分区：
文献类型：
--
作者：
Edwards TG;Vidmar TJ;Koeller K;Bashkin JK;Fisher C
本文报道了控制 HPV 游离 DNA 稳定性的 DNA 损伤反应 (DDR) 基因和途径。我们着手了解 DNA 结合的 N-甲基吡咯-咪唑发夹聚酰胺 (PA25) 导致细胞中 HPV DNA 大量丢失的机制。 Southern 印迹显示 PA25 在治疗 5 小时内改变 HPV 游离体。基因表达阵列鉴定出许多 DDR 基因在含有 HPV16 附加体的细胞 (W12E) 中被 PA25 特异性改变，但在 HPV 阴性 (C33A) 细胞或整合 HPV16 的细胞 (SiHa) 中则没有。然后对 240 个 DDR 基因进行 siRNA 筛选，以确定 PA25 活性的增强子和阻遏子。偶然地，筛选还发现了许多新基因，例如 TDP1 和 TDP2，调节正常 HPV 附加体的稳定性。 MRN 和 9-1-1 复合物对于 PA25 介导的附加体破坏非常重要，并被选择用于后续研究。 Mre11 与其他同源重组和 dsDNA 断裂修复基因一起，属于高度显着的 PA25 阻遏基因。研究发现 Mre11 抑制剂 Mirin 可使 HPV 附加体对 PA25 敏感，导致 PA25 IC50 降低约 5 倍。一种将 DNA 末端标记与 Q-PCR 结合的新测定表明，PA25 会导致 HPV DNA 内的链断裂，而味醂可大大增强这种活性。 9-1-1 复合体成员 Rad9 是一种代表性的 PA25 增强子，在 PA25 处理后短暂磷酸化，表明它在检测 PA25 对细胞造成的附加体损伤并发出信号中发挥作用。这些结果证实 DNA 靶向化合物进入细胞并特异性靶向 HPV 附加体。这一作用导致众多 DDR 途径的激活以及细胞中游离 DNA 的大量消除。我们的研究结果表明，病毒游离体可以通过小沟结合剂从细胞中消除，并表明 DDR 途径是该过程的重要介质。
DNA damage response (DDR) genes and pathways controlling the stability of HPV episomal DNA are reported here. We set out to understand the mechanism by which a DNA-binding, N-methylpyrrole-imidazole hairpin polyamide (PA25) acts to cause the dramatic loss of HPV DNA from cells. Southern blots revealed that PA25 alters HPV episomes within 5 hours of treatment. Gene expression arrays identified numerous DDR genes that were specifically altered in HPV16 episome-containing cells (W12E) by PA25, but not in HPV-negative (C33A) cells or in cells with integrated HPV16 (SiHa). A siRNA screen of 240 DDR genes was then conducted to identify enhancers and repressors of PA25 activity. Serendipitously, the screen also identified many novel genes, such as TDP1 and TDP2, regulating normal HPV episome stability. MRN and 9-1-1 complexes emerged as important for PA25-mediated episome destruction and were selected for follow-up studies. Mre11, along with other homologous recombination and dsDNA break repair genes, was among the highly significant PA25 repressors. The Mre11 inhibitor Mirin was found to sensitize HPV episomes to PA25 resulting in a ∼5-fold reduction of the PA25 IC50. A novel assay that couples end-labeling of DNA to Q-PCR showed that PA25 causes strand breaks within HPV DNA, and that Mirin greatly enhances this activity. The 9-1-1 complex member Rad9, a representative PA25 enhancer, was transiently phosphorylated in response to PA25 treatment suggesting that it has a role in detecting and signaling episome damage by PA25 to the cell. These results establish that DNA-targeted compounds enter cells and specifically target the HPV episome. This action leads to the activation of numerous DDR pathways and the massive elimination of episomal DNA from cells. Our findings demonstrate that viral episomes can be targeted for elimination from cells by minor groove binding agents, and implicate DDR pathways as important mediators of this process.
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