| Human topoisomefase I (Top1) is an essential nuclear protein. It is also the target of the antineoplastic agent camptothecin. In order to gain knowledge regarding the cellular function of Top1, as well as on the mechanism of camptothecin cytotoxicity, we sought to identify Top1 binding proteins. To accomplish this aim, we employed a modified two hybrid screen in S. cerevisiae using the amino terminus of Top1 (Top1 a, amino acids 1–250) fused to the Gal4 DNA binding domain (BD). Candidate Top1 binding proteins were then verified as true positives by several methods, including coimmunoprecipitation and affinity chromatography. During these confirmatory studies, a serendipitous finding was made that SV40 Large T Antigen (Tag) binds to Top1a. After demonstrating that Tag could bind Top1a in vitro and in vivo, we then determined that Tag has two distinct binding sites on Top1: one within amino acids 1–139 and another within the carboxy terminus (amino acids 383–765). The finding that Tag binds Top1 is consistent with other reports of DNA helicase-topoisomerase interactions. Indeed, the finding that Tag and Top1 physically interact was confirmed by another group during the course of this work. Among the several putative Top1a-binding proteins identified by the two-hybrid screen, we have focused on characterizing a novel gene product that contains a region rich in arginine/serine (RS) repeats. This protein, denoted Topors, is recognized by mAb104 and is phosphorylated by SRPK1, confirming that it belongs to the SR family of proteins. However, unlike many SR proteins, Topors lacks a recognizable RNA recognition motif (RRM). Cellular localization studies indicate that Topors localizes to subnuclear domains characteristic of splicing and transcription proteins, suggesting that Topors functions in one or both of these processes. The finding that Topors interacts with Top1 is consistent with roles for Top1 in transcription and mRNA splicing. |
