TRANSPOSASES IN ETOPOSIDE RESISTANCE

描述(申请人提供)：转座酶活性在人类中被认为已经灭绝，因为DNA运动在高等生物中可能是有害的，导致基因组不稳定，甚至可能是恶性的。然而，我们分离到一种名为Metnase的人类转座酶蛋白，它具有组蛋白甲基酶和非同源末端连接(NHEJ)DNA修复活性。发现它与DNA连接酶IV相互作用，与其NHEJ修复活性一致。Metnase也是一种对超螺旋DNA有选择性的内切酶。因此，我们探索了Metnase在破坏复制的染色单体中的作用。金属酶与DNA降解的关键酶拓扑异构酶II(Topo II)相互作用，在体外和细胞内均能增强其DNA降解活性。Metnase转座酶结构域中的核酸酶活性是充分增强Topo II杀菌活性所必需的。Metnase提高了Topo II降解DNA的速度，并增强了Topo II抵抗降解抑制剂ICRF-193的能力。Metnase改善了Topo II对ICRF-193的耐药性，这一发现促使人们研究它是否可以介导对临床相关Topo II抑制剂依托泊苷的耐药性。我们发现Metnase在体外阻止了依托泊苷对Topo II降解的抑制，并介导了细胞对依托泊苷的抵抗，在依托泊苷存在的情况下促进了增殖。Metnase还促进依托泊苷诱导的DSB更快地清除。因此，Metnase似乎介导了对依托泊苷诱导的DNA损伤和细胞周期停滞的抵抗。这是一种尚未被探索的新的依托泊苷耐药机制。本申请建议通过提出三个问题来确定Metnase介导依托泊苷耐药的机制：1)调节Metnase还原依托泊苷DSB能力的上游信号是什么？2)Metnase还原依托泊苷DSB的下游途径是什么？3)Metnase水平是否预示着人类恶性肿瘤对依托泊苷的临床耐药？与公共健康相关：我们已经分离出一种名为Metnase的新蛋白质，它可以帮助染色体修复断裂并解开缠结，从而使它们在细胞分裂期间正确分离。由于这些作用，Metnase介导了抗癌药物依托泊苷的耐药性。了解Metnase做到这一点的机制将允许产生针对Metnase的药物，这可能有助于改善癌症患者对依托泊苷的反应。此外，Metnase水平可以预测哪个患者会对依托泊苷有反应。

DESCRIPTION (provided by applicant): Transposase activity was thought to be extinct in humans because DNA movement can be deleterious in higher organisms, resulting in genomic instability and perhaps malignancy. However, we isolated a human transposase protein termed Metnase that had histone methylase and non-homologous end-joining (NHEJ) DNA repair activity. It was found to interact with DNA Ligase IV, consistent with its NHEJ repair activity. Metnase also was an endonuclease preferential for supercoiled DNA. We therefore explored Metnase's role in decatenating replicated chromatids. Metnase interacted with Topoisomerase II (Topo II ), the critical decatenating enzyme, and enhanced its activity in DNA decatenation, both in vitro and intracellularly. The nuclease activity within the transposase domain of Metnase was required for full enhancement of Topo II decatenating activity. Metnase improved the rate at which Topo II decatenated DNA, and increased the ability of Topo II to resist the decatenation inhibitor ICRF-193. The finding that Metnase improved Topo II resistance to ICRF-193 stimulated an investigation into whether it could mediate resistance to the clinically relevant Topo II inhibitor etoposide. We found that Metnase prevented inhibition of Topo II decatenation by etoposide in vitro, and mediated cellular resistance to etoposide, promoting proliferation in the presence of etoposide. Metnase also promoted a more rapid clearance of etoposide-induced DSB. Thus, Metnase appeared to mediate resistance to etoposide-induced DNA damage and cell cycle arrest. This is a novel mechanism of etoposide resistance that is unexplored. This application proposes to define the mechanism by which Metnase mediates resistance to etoposide by asking three questions: 1) What are the upstream signals that regulate the ability of Metnase to reduce etoposide DSBs? 2) What is the downstream pathway by which Metnase reduces etoposide DSBs? 3) Do Metnase levels predic clinical resistance to etoposide in human malignancy? PUBLIC HEALTH RELEVANCE: We have isolated a novel protein termed Metnase that helps chromosomes repair breaks and also untangle, thereby allowing them to separate properly during cell division. Because of these actions, Metnase mediates resistance to the cancer drug etoposide. Understanding the mechanism by which Metnase does this would allow the generation of drugs targeting Metnase, which could help improve the response of cancer patients to etoposide. In addition, the levels of Metnase could predict which patient will respond to etoposide.