Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SRprotein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression. DNA topoisomerase I (topo I)2 is a constitutively expressed multifunctional enzyme that localizes at active transcription sites (1, 2). Its best known function is to control the topological state of DNA by relieving torsional stress that is generated following DNA strand separation during transcription, replication, and repair (3-5). The catalytic mechanism involves the formation of a DNA⅐topo I complex (cleavage complex) with the enzyme being covalently bound to the 3Ј-end of the cleaved DNA strand through a tyrosine-phosphate ester bond. Cleavage complexes are usually short-lived; their stabilization by compounds of the camptothecin (CPT) family of anticancer drugs may cause DNA strand break accumulation and eventually lead to cell death. Human topo I can relax both negative and positive supercoils by controlled rotation of the DNA strand downstream of the cleavage site followed by break resealing and restoration of an intact DNA duplex.In addition to relaxing supercoiled DNA, human topo I also plays a major role in pre-mRNA splicing, being endowed with a protein kinase activity targeted at a group of splicing factors of the serine-arginine (SR)-rich protein family (6). SR-proteins function both as components of the basal RNA splicing machinery and as regulators of alternative splicing (7, 8). Moreover, SR-proteins are involved in the control of mRNA transport and stability (8, 9) and contribute to the maintenance of genomic stability (10). SR-proteins are structurally characterized ...

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BackgroundLow cellular level of BID is critical for viability of numerous cancer cells. Sensitization of cells to anticancer agents by BID overexpression from adenovirus or pcDNA vectors is a proposed strategy for cancer therapy; however it does not provide any stringent control of cellular level of BID. The aim of this work was to examine whether a fusion of BID with TAT cell penetrating peptide (TAT-BID) may be used for controlled sensitization of cancer cells to anticancer agents acting through death receptors (TRAIL) or DNA damage (camptothecin). Prostate cancer PC3 and LNCaP, non-small human lung cancer A549, and cervix carcinoma HeLa cells were used in the study.MethodsUptake of TAT-BID protein by cells was studied by quantitative Western blot analysis of cells extracts. Cells viability was monitored by MTT test. Apoptosis was detected by flow cytometry and cytochrome c release assay.ResultsTAT-BID was delivered to all cancer cells in amounts depending on time, dose and the cell line. Recombinant BID sensitized PC3 cells to TRAIL or, to lesser extent, to camptothecin. Out of remaining cells, TAT-BID sensitized A549, and only slightly HeLa cells to TRAIL. None of the latter cell lines were sensitized to camptothecin. In all cases the mutant not phosphorylable by CK2 (TAT-BIDT59AS76A) was similarly efficient in sensitization as the wild type TAT-BID.ConclusionsTAT-BID may be delivered to cancer cells in controlled manner and efficiently sensitizes PC3 and A549 cells to TRAIL. Therefore, it may be considered as a potential therapeutic agent that enhances the efficacy of TRAIL for the treatment of prostate and non-small human lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-771) contains supplementary material, which is available to authorized users.

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Human topoisomerase I is partitioned between the nucleolus and the nucleoplasm in the interphase cells. Under unstressed conditions it is concentrated in the first compartment but nucleolar concentration of the full length protein is lost after inactivation of relaxation activity. Due to the above, subnuclear localization of topoisomerase I is linked with DNA relaxation activity of topoisomerase I. Looking for other factors responsible for subnuclear distribution of topoisomerase I, we studied here localization of the fluorescently tagged fragments and point mutants of topoisomerase I in HeLa cells. We found that two regions of topoisomerase I, the N-terminal and the linker domains, were critical for subnuclear localization of the enzyme. The linker domain and the distal region of the N-terminal domain directed topoisomerase I to the nucleolus, whereas the remaining region of the N-terminal domain was responsible for the nucleoplasmic localization. The effects exhibited by the regions which contributed to nuclear distribution of topoisomerase I were independent of DNA relaxation activity. Localization mutations in both domains complemented one another giving the wild-type phenotype for the double mutant. These results suggest a two-stage model of regulation of partitioning of topoisomerase I between the nucleolus and the nucleoplasm. The first stage is a net of interactions provided by the N-terminal and the linker domains. The other stage, accessible only if the first net is balanced, is driven by DNA relaxation activity. J. Cell. Biochem. 118: 407-419, 2017. © 2016 Wiley Periodicals, Inc.