The p14ARF Alternate Reading Frame Protein Enhances DNA Binding of Topoisomerase I by Interacting with the Serine 506-Phosphorylated Core Domain

In addition to its well-characterized function as a tumor suppressor, p14ARF (ARF) is a positive regulator of topoisomerase I (topo I), a central enzyme in DNA metabolism and a target for cancer therapy. We previously showed that topo I hyperphosphorylation, a cancer-associated event mediated by elevated levels of the protein kinase CK2, increases topo I activity and the cellular sensitivity to topo I-targeted drugs. Topo I hyperphosphorylation also increases its interaction with ARF. Because the ARF−topo I interaction could be highly relevant to DNA metabolism and cancer treatment, we identified the regions of topo I involved in ARF binding and characterized the effects of ARF binding on topo I function. Using a series of topo I deletion constructs, we found that ARF interacted with the topo I core domain, which encompasses most of the catalytic and DNA-interacting residues. ARF binding increased the DNA relaxation activity of hyperphosphorylated topo I by enhancing its association with DNA, but did not affect the topo I catalytic rate. In cells, ARF promoted the chromatin association of hyperphosphorylated, but not basal phosphorylated, topo I, and increased topo I-mediated DNA nicking under conditions of oxidative stress. The aberrant nicking was found to correlate with increased formation of DNA double-strand breaks, which are precursors of many genome destabilizing events. The results suggest that the convergent actions of oxidative stress and elevated CK2 and ARF levels, which are common features of cancer cells, lead to a dysregulation of topo I that may contribute both to the cellular response to topo I-targeted drugs and to genome instability.

p14ARF（ARF）除了作为功能已得到充分阐明的肿瘤抑制因子外，同时还是拓扑异构酶I（topoisomerase I，topo I）的正向调控因子——拓扑异构酶I是DNA代谢过程中的核心酶，也是癌症治疗的重要靶点。我们此前的研究表明，由蛋白激酶CK2水平升高介导的、与癌症相关的拓扑异构酶I过度磷酸化事件，可增强拓扑异构酶I的活性，以及细胞对拓扑异构酶I靶向药物的敏感性。此外，拓扑异构酶I的过度磷酸化还会增强其与ARF的相互作用。鉴于ARF与拓扑异构酶I的相互作用可能与DNA代谢及癌症治疗密切相关，本研究鉴定出了拓扑异构酶I中参与ARF结合的区域，并解析了ARF结合对拓扑异构酶I功能的影响。通过一系列拓扑异构酶I缺失突变体实验，我们发现ARF可与拓扑异构酶I的核心结构域结合，该结构域包含了绝大多数催化位点及DNA结合残基。ARF结合可通过增强过度磷酸化拓扑异构酶I与DNA的结合能力，提升其DNA松弛活性，但并不会改变拓扑异构酶I的催化速率。在细胞实验中，ARF可促进过度磷酸化（而非基础磷酸化状态）的拓扑异构酶I与染色质的结合，并在氧化应激条件下增强拓扑异构酶I介导的DNA单链切口形成。研究发现，这种异常的单链切口形成与DNA双链断裂的形成增多相关，而DNA双链断裂是诸多基因组不稳定事件的前驱诱因。上述研究结果表明，氧化应激、蛋白激酶CK2与ARF水平升高（这些均为癌细胞的常见特征）的协同作用，会导致拓扑异构酶I的功能失调，这既可能影响细胞对拓扑异构酶I靶向药物的应答，也可能促进基因组不稳定的发生。