Interaction between human topoisomerase I and a novel RING finger/arginine-serine protein

Paul Haluska Jr 0 Ahamed Saleem 0 Zeshaan Rasheed 0 Farheena Ahmed 0 Emily W. Su 0 Leroy F. Liu 0 Eric H. Rubin 0 0 Departments of Pharmacology and Medicine, 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 The N-terminus of human topoisomerase I participates in the binding of this enzyme to helicases and other proteins. Using the N-terminal 250 amino acids of human topoisomerase I and a yeast two-hybrid/ in vitro binding screen, a novel arginine-serine-rich peptide was identified as a human topoisomerase Ibinding protein. The corresponding full-length protein, named topors, contains a consensus RING zinc finger domain and nuclear localization signals in addition to the arginine-serine-rich region. The RING finger domain of topors is homologous to a similar domain in a family of viral proteins that are involved in the regulation of viral transcription. When expressed in HeLa cells as a green fluorescent protein fusion, topors localizes in the nucleus in a punctate pattern and co-immunoprecipitates with topoisomerase I. These data suggest that topors is involved in transcription, possibly recruiting topoisomerase I to RNA polymerase II transcriptional complexes. - Human DNA topoisomerase I (htop1) is a 100 kDa monomeric nuclear protein that regulates DNA topology and is the target of an important new class of antineoplastic compounds, the camptothecins (1). Although proliferating mammalian cells require top1 for viability (2), five mammalian topoisomerases have been identified to date (3) and the functions of mammalian top1 that are indispensable are not yet known (4). Furthermore, relatively little is known regarding interactions between htop1 and other proteins, although these interactions are likely to be important in both the cellular function of htop1 and in the cytotoxic mechanisms of camptothecins. Indeed, physical interactions between htop1 and the TATA-binding protein (5) and between htop1 and nucleolin (6) may underlie certain roles of top1 in RNA polymerase II-mediated (5,7,8) and RNA polymerase I-mediated (9) transcription, respectively. Moreover, the effects of camptothecin may be modulated by a physical interaction between htop1 and the SV40 T antigen helicase (10). Results from our laboratory and others indicate that interactions between htop1 and other proteins are mediated at least in part by the N-terminal 210 amino acids of htop1, which are dispensable for DNA topoisomerase activity (6,11,12). Therefore, to identify other htop1-binding proteins that may be important in the function of htop1 and in camptothecinmediated cytotoxicity, we established a yeast two-hybrid/in vitro binding screen using the N-terminus of htop1. We now report the results of this screen and the identification of a novel nuclear protein that contains a RING finger and an arginineserine (RS)-rich domain. MATERIALS AND METHODS The pAS2-Atop1 vector was used to express amino acids 2250 of htop1 (Atop1) linked to the C-terminus of a GAL4 DNAbinding domain (BD) peptide in Saccharomyces cerevisiae strain Y199 containing a 2 m M plasmid-based b -galactosidase reporter system and a HIS3-based reporter system as described (13). Using this strain and a lithium acetate method, a HeLa cDNA library (Human HeLa S3 Matchmaker cDNA library; Clontech) expressing GAL4 activation domain (AD) fusion proteins was co-transformed with the pAS2-Atop1 vector; transformants were selected by the use of appropriate media. Histidine prototrophs were tested for b -galactosidase activity using a filter lift technique and 5bromo-4-chloro-3-indolyl-b -D-galactopyranoside as described (14). Colonies that expressed b -galactosidase were subsequently grown on plates containing 10 m g/ml cycloheximide to select for loss of the pAS2-Atop1 plasmid, which contains the dominant cycloheximide sensitivity marker, CYH2 (15). Since the pAS2Atop1 plasmid also contains a TRP marker, loss of the plasmid was confirmed by assays for growth on media lacking tryptophan. The resultant colonies, containing HeLa cDNAs encoding putative htop1-binding proteins, were tested in a secondary mating screen described next. To develop a secondary screen for clones that scored positive in the htop1-baited two-hybrid assay, yeast strain Y187 (MATa ; Clontech) was transformed with plasmids expressing either the BD alone, BDlamin C, BDJak1 (kindly provided by Brian Pollack and Sidney Petska, UMDNJ, Piscataway, NJ) or BDAtop1. These strains were streaked in vertical lines on YPD plates, with Y199 (MATa) strains containing putative top1-binding proteins streaked in horizontal lines. After 12 days growth, diploids were selected by replica plating to plates lacking both leucine and tryptophan. After an additional 5 days growth, b -galactosidase expression in the diploid colonies was assayed as described above. In vitro top1-binding assay A GST fusion protein containing amino acids 2250 of htop1 was covalently linked to glutathioneSepharose beads using the bifunctional imidoester dimethylpimelimidate dihydrochloride as described (6). Yeast extracts were prepared by lysis in ice-cold RIPA buffer (50 mM TrisHCl, pH 7.2, 150 mM NaCl, 0.1% SDS, 1% Triton X-100 and 1% Na deoxycholate) with protease inhibitors (1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.5 m g/ml leupeptin and 1 m g/ml pepstatin) and binding assays performed in 1 ml PBS, 0.2% Tween-20 containing protease inhibitors as described (13). Proteins remaining bound to the htop1-linked beads were visualized by immunoblotting with a monoclonal antibody recognizing the AD (Clontech) and an enhanced chemiluminescent technique as described (6). RNA isolation and northern blot hybridization Total RNA was isolated from U-937 human monoblastic leukemia cells using guanidine isothiocyanate/cesium chloride gradient centrifugation as described (16). Poly(A)+ RNA was purified from total RNA using an oligo(dT) cellulose column as described (17). The RNA was separated by electrophoresis in 1% agarose, 2.2 M formaldehyde gels and transferred to nitrocellulose filters. A 32P-labeled DNA probe was generated by digestion of the 182 HeLa cDNA library clone with EcoRI and XhoI, followed by labeling of the resultant 1.3 kb insert using random hexamers, [32P]dCTP and the Klenow DNA polymerase. Hybridizations were performed as described (16). The filters were washed and exposed to Kodak X-Omat XAR film using an intensifying screen. Topors cDNA isolation and sequencing An oligonucleotide designed to anneal to the mRNA 5'-cap region (SMART oligo; Clontech) was used with U-937 RNA to obtain topors cDNA 5'-sequence relative to the original fragment identified in the HeLa two-hybrid cDNA library clone. A human fetal lung cDNA library (Clontech) and PCR were also used to obtain additional topors cDNA 5'- and 3'fragments. Sequencing was performed using an ABI Prism 377 automated DNA sequencer (Perkin-Elmer). Analyses o (...truncated)