Translocated in liposarcoma (TLS) is an importantprotein component of the heterogeneous nuclear ribonucleoprotein complex involved in the splicing of pre-mRNA and the export of fully processed mRNA to the cytoplasm. We examined the domain organization of human TLS by a combined approach using limited proteolysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, circular dichroism, inductively coupled plasma atomic emission spectroscopy, and NMR spectroscopy. We found that the RNA recognition motif (RRM) and zinc finger-like domains exclusively form protease-resistant core structures within the isolated TLS protein fragments, while the remaining regions, including the Arg-Gly-Gly repeats, appear to be completely unstructured. Thus, TLS contains the unstructured N-terminal half followed by the RRM and zinc finger-like domains, which are connected to each other by a flexible linker. We also carried out NMR analyses to obtain more detailed insights into the individual RRM and zinc finger-like domains. The 113 Cd NMR analysis of the zinc finger-like domain verified that zinc is coordinated with four cysteines in the C4 type scheme. We also investigated the interaction of each domain with an oligo-RNA containing the GGUG sequence, which appears to be critical for the TLS function in splicing. The backbone amide NMR chemical shift perturbation analyses indicated that the zinc finger domain binds GGUG-containing RNA with a dissociation constant of about 1.0 ؋ 10 ؊5 M, whereas the RRM domain showed no observable interaction with this RNA. This surprising result implies that the zinc finger domain plays a more predominant role in RNA recognition than the RRM domain.The translocated in liposarcoma (TLS) 1 protein, also termed FUS, was first identified in human myxoid and round cell liposarcomas as an oncogenic fusion protein with a stressinduced DNA-binding transcription factor, CCAAT enhancerbinding homologous protein (CHOP, also known as GADD153 or DDIT3) (1, 2). The resultant fusion protein (TLS-CHOP), consisting of the N-terminal half of TLS and the full-length CHOP, appears to act as a potent transcription factor possibly by combining the TLS transactivation activity and the CHOP DNA binding activity. A different type of fusion protein, TLS-ERG (a member of the erythroblast transformation-specific (ETS) family of transcription factors), was subsequently detected in human acute myeloid leukemia (3).The normal TLS, consisting of 526 amino acids with a calculated molecular mass of 53 kDa, belongs to a family including the closely related proteins Ewing's sarcoma (EWS) (4) and TAF II 68 (TATA-binding protein-associated factor) (5). Thus, they are collectively called the TET (TLS, EWS, TAF II 68) family. EWS and TAF II 68 interact with components of the RNA polymerase II complex (5, 6). Moreover sarcoma-associated RNA-binding fly homologue (SARFH), a Drosophila homologue of TLS, is colocalized with the polymerase on active chromatin (7). The N-terminal domain of TLS is involved in transcription...
A novel DNA polymerase, designated as OsPolI-like, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare). The OsPolI-like cDNA was 3765 bp in length, and the open reading frame encoded a predicted product of 977 amino acid residues with a molecular weight of 100 kDa. The OsPolI-like gene has been mapped to chromosome 8 and contains 12 exons and 11 introns. The encoded protein showed a high degree of sequence and structural homology to Escherichia coli pol I protein, but differed from DNA polymerase gamma and theta. The DNA polymerase domain of OsPolI-like showed DNA polymerase activity. Subcellular fractionation analysis suggested that the protein is localized in the plastid. Northern and western blotting, and in situ hybridization analyses demonstrated preferential expression of OsPolI-like in meristematic tissues such as shoot apical meristem, root apical meristem, leaf primordia and the marginal meristem. Interestingly, no expression was detected in mature leaves, although they have a high chloroplast content. These properties indicated that OsPolI-like is a novel plant DNA polymerase. The function of OsPolI-like is discussed in relation to plastid maturation.
Petasiphenol, a bio-antimutagen isolated from a Japanese vegetable, Petasites japonicus, selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta, and epsilon but also showed no effect even on the pol beta activity, the three-dimensional structure of which is thought to be highly similar to pol lambda. The inhibitory effect of petasiphenol on intact pol lambda including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50% inhibition was observed at a concentration of 7.8 microM. The petasiphenol-induced inhibition of the pol lambda activity was noncompetitive with respect to both the DNA template-primer and the dNTP substrate. Petasiphenol did not only inhibit the activity of the truncated pol lambda including the pol beta-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of pol lambda but did not bind to the C-terminal region. On the basis of these results, the pol lambda inhibitory mechanism of petasiphenol is discussed.
Eukaryotic cells reportedly contain three replicative DNAdirected DNA polymerases (EC 2.7.7.7) (pol ␣, ␦, and ⑀), 1 mitochondrial DNA polymerase (pol ␥), and at least nine repair types of DNA polymerase (pol , ␦, ⑀, , , , , , and ) (1-8), and we have screened for natural compounds that selectively inhibit each of these eukaryotic DNA polymerases and found several inhibitors (9 -15). The purpose was to use the compounds as tools and molecular probes to distinguish DNA polymerases and to clarify their biological and in vivo functions. In this study, we found an interesting fungus-produced compound that selectively inhibits the activities of pol  and pol . Interestingly, the compound was found to be a plant phytotoxin, solanapyrone A, isolated from the causal fungus of early blight disease in potato. To our knowledge, such specific natural compounds have not been reported with the exception of the pol -inhibitor, prunasin, which we reported previously (13). The compound was a stronger pol -inhibitor than prunasin, and no pol -inhibitors have been reported.Pol  is the lowest molecular mass (39 kDa) DNA polymerase lacking such intrinsic accessory activities as 3Ј-or 5Ј-exonuclease, endonuclease, dNMP turnover, or pyrophosphorolysis (16). Pol  consists of an independently folded N-terminal 8-kDa domain and C-terminal 31-kDa domain (17,18). The N-terminal domain was originally characterized as a single-stranded DNA (ssDNA)-binding domain. It was then found to possess binding specificity for the 5Ј-phosphate in gapped DNA (19 -21) and a helix-hairpin-helix motif found in several other DNA repair enzymes (22,23). Recently, Matsumoto and Kim (24) demonstrated that pol  catalyzes the removal of dRP from the AP endonuclease-incised AP site via -elimination as opposed to hydrolysis, and that this dRP lyase activity resides in the 8-kDa domain of pol . According to recent studies (7, 25), moreover, pol  is known to have another family polymerase, pol . Pol  has been present in all tissues examined and is generally expressed at a low level as are a number of other so-called constitutive "house-keeping" enzymes. Although the in vivo function of the family enzyme, pol , is unclear as yet, pol appears to work in a similar manner to pol  (7). As to why a plant phytotoxin is a pol -family-specific inhibitor, we presently are analyzing the structure and function of pol  and pol using the inhibitor from two different view-points to understand the precise role of each of the polymerases in vivo and to develop a drug design strategy for cancer chemotherapy agents. We previously reported the three-dimensional structure of pol  and the N-terminal 8-kDa domain (the DNA template-binding domain) with or without long chain fatty acids to further clarify the structure and function of pol  (26). Because at least pol  is an essential enzyme for nucleotide excision repair (1, 2), the plant phytotoxin may lead to blockage of the DNA repair systems of rescue cancer cells under clinical radiation-therapy or chemotherapy. The plan...
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