Departments of Medicine and Pathology, Herbert Irving Comprehensive Cancer Center, Columbia University, 177 Ft. Washington Avenue, MHB 6N-435, New York, NY 10032, USA.
Biochem J. 2013 Jul 1;453(1):125-36. doi: 10.1042/BJ20130012.
NKX3.1 (NK3 homeobox 1) is a prostate tumour suppressor protein with a number of activities that are critical for its role in tumour suppression. NKX3.1 mediates the cellular response to DNA damage by interacting with ATM (ataxia telangiectasia mutated) and by activation of topoisomerase I. In the present study we characterized the interaction between NKX3.1 and topoisomerase I. The NKX3.1 homeodomain binds to a region of topoisomerase I spanning the junction between the core and linker domains. Loss of the topoisomerase I N-terminal domain, a region for frequent protein interactions, did not affect binding to NKX3.1 as was shown by the activation of Topo70 (N-terminal truncated topoisomerase I) in vitro. In contrast, NKX3.1 interacts with the enzyme reconstituted from peptide fragments of the core and linker active site domains, but inhibits the DNA-resolving activity of the reconstituted enzyme in vitro. The effect of NKX3.1 on both Topo70 and the reconstituted enzyme was seen in the presence and absence of camptothecin. Neither NKX3.1 nor CPT (camptothecin) had an effect on the interaction of the other with topoisomerase I. Therefore the interactions of NKX3.1 and CPT with the linker domain of topoisomerase I are mutually exclusive. However, in cells the effect of NKX3.1 on topoisomerase binding to DNA sensitized the cells to cellular toxicity and the induction of apoptosis by low doses of CPT. Lastly, topoisomerase I is important for the effect of NKX3.1 on cell survival after DNA damage as topoisomerase knockdown blocked the effect of NKX3.1 on clonogenicity after DNA damage. Therefore NKX3.1 and topoisomerase I interact in vitro and in cells to affect the CPT sensitivity and DNA-repair functions of NKX3.1.
NKX3.1（NK3 同源盒 1）是一种前列腺肿瘤抑制蛋白，具有许多关键的肿瘤抑制作用。NKX3.1 通过与 ATM（共济失调毛细血管扩张突变）相互作用和激活拓扑异构酶 I 来介导细胞对 DNA 损伤的反应。在本研究中，我们对 NKX3.1 与拓扑异构酶 I 之间的相互作用进行了表征。NKX3.1 同源结构域与拓扑异构酶 I 的核心和连接子结构域之间的连接区结合。拓扑异构酶 I 的 N 端结构域缺失，这是一个频繁发生蛋白相互作用的区域，不会影响与 NKX3.1 的结合，这一点从体外激活 Topo70（N 端截断的拓扑异构酶 I）中可以看出。相反，NKX3.1 与核心和连接子活性位点结构域的肽片段重建的酶相互作用，但在体外抑制重建酶的 DNA 解析活性。在 Camptothecin（CPT）存在和不存在的情况下，NKX3.1 对 Topo70 和重建酶都有影响。NKX3.1 和 CPT 都不会影响彼此与拓扑异构酶 I 的相互作用。因此，NKX3.1 和 CPT 与拓扑异构酶 I 的连接子结构域的相互作用是相互排斥的。然而，在细胞中，NKX3.1 对拓扑异构酶与 DNA 结合的影响使细胞对低剂量 CPT 的细胞毒性和凋亡诱导敏感。最后，拓扑异构酶 I 对 NKX3.1 对 DNA 损伤后细胞存活的影响很重要，因为拓扑异构酶敲低阻断了 NKX3.1 对 DNA 损伤后集落形成能力的影响。因此，NKX3.1 和拓扑异构酶 I 在体外和细胞内相互作用，影响 CPT 敏感性和 NKX3.1 的 DNA 修复功能。
