Haluska P, Saleem A, Edwards T K, Rubin E H
Department of Pharmacology, Robert Wood Johnson Medical School and the Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08901, USA.
Nucleic Acids Res. 1998 Apr 1;26(7):1841-7. doi: 10.1093/nar/26.7.1841.
We have attempted to identify human topoisomerase I-binding proteins in order to gain information regarding the cellular roles of this protein and the cytotoxic mechanisms of the anticancer drug camptothecin, which specifically targets topoisomerase I. In the course of this work we identified an interaction between the N-terminus of human topoisomerase I and the SV40 T antigen that is detectable in vitro using both affinity chromatography and co-immunoprecipitation. Additional results indicate that this interaction does not require intermediary DNA or stoichiometric quantities of other proteins. Furthermore, the interaction is detectable in vivo using a yeast two-hybrid assay. Two binding sites for T antigen are apparent on the topoisomerase I protein: one consisting of amino acids 1-139, the other present in the 383-765 region of the protein. Interestingly, nucleolin, which binds the 166-210 region of topoisomerase I, is able to bind an N-terminal fragment of topoisomerase I concurrently with T antigen. Taken together with our prior identification of nucleolin as a topoisomerase I-binding protein, the current results suggest that helicase-binding is a major role of the N-terminus of human topoisomerase I and that the resultant helicase-topoisomerase complex may function as a eukaryotic gyrase.
我们试图鉴定人拓扑异构酶I结合蛋白，以便获取有关该蛋白的细胞作用以及抗癌药物喜树碱（其特异性靶向拓扑异构酶I）的细胞毒性机制的信息。在这项工作过程中，我们鉴定出人类拓扑异构酶I的N端与SV40 T抗原之间存在相互作用，这种相互作用在体外利用亲和层析和共免疫沉淀法均可检测到。其他结果表明，这种相互作用不需要中间DNA或化学计量的其他蛋白质。此外，利用酵母双杂交试验在体内也可检测到这种相互作用。在拓扑异构酶I蛋白上有两个明显的T抗原结合位点：一个由氨基酸1 - 139组成，另一个存在于该蛋白的383 - 765区域。有趣的是，结合拓扑异构酶I的166 - 210区域的核仁素能够与T抗原同时结合拓扑异构酶I的一个N端片段。结合我们之前将核仁素鉴定为拓扑异构酶I结合蛋白的结果，目前的结果表明解旋酶结合是人类拓扑异构酶I N端的主要作用，并且由此产生的解旋酶 - 拓扑异构酶复合物可能发挥真核生物促旋酶的功能。
