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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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.

Isolation and detection of circulating tumor cells (CTCs) from human blood plays an important role in non- invasive screening of cancer evolution and in predictive therapeutic treatment. Here, we present the novel tool utilizing: (i) the microfluidic device with (ii) incorporated photovoltaic (PV) based SERS-active platform, and (iii) shell-isolated nanoparticles (SHINs) for simultaneous separation and label-free analysis of circulating tumour cells CTCs in the blood specimens with high specificity and sensitivity. The proposed microfluidic chip enables the efficient size – based inertial separation of circulating cancer cells from the whole blood samples. The SERS-active platform incorporated into the microfluidic device permits the label-free detection and identification of isolated cells through the insight into their molecular and biochemical structure. Additionally, the silver nanoparticles coated with an ultrathin shell of silica (Ag@SiO 2 ) was used to improve the detection accuracy and sensitivity of analysed tumor cells via taking advantages of shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS). The empirical analysis of SHINERS spectra revealed that there are some differences among studied (HeLa), renal cell carcinoma (Caki-1), and blood cells. Unique SHINERS features and differences in bands intensities between healthy and cancer cells might be associated with the variations in the quantity and quality of molecules such as lipid, protein, and DNA or their structure during the metastasis cancer formation. To demonstrate the statistical efficiency of the developed method and improve the differentiation for circulating tumors cells detection the principal component analysis (PCA) has been performed for all SHINERS data. PCA method has been applied to recognize the most significant differences in SHINERS data among the three analyzed cells: Caki-1, HeLa, and blood cells. The proposed approach challenges the current multi-steps CTCs detection methods in the terms of simplicity, sensitivity, invasiveness, destructivity, time and cost of analysis, and also prevents the defragmentation/damage of tumor cells and thus leads to improving the accuracy of analysis. The results of this research work show the potential of developed SERS based tool for the separation of tumor cells from whole blood samples in a simple and minimally invasive manner, their detection and molecular characterization using one single technology.

The detection and monitoring of circulating tumor cells (CTCs) in blood is an important strategy for early cancer evidence, analysis, monitoring of therapeutic response, and optimization of cancer therapy treatments. In this work, tailor-made membranes (MBSP) for surface-enhanced Raman spectroscopy (SERS)-based analysis, which permitted the separation and enrichment of CTCs from blood samples, were developed. A thin layer of SERS-active metals deposited on polymer mat enhanced the Raman signals of CTCs and provided further insight into CTCs molecular and biochemical composition. The SERS spectra of all studied cells—prostate cancer (PC3), cervical carcinoma (HeLa), and leucocytes as an example of healthy (normal) cell—revealed significant differences in both the band positions and/or their relative intensities. The multivariate statistical technique based on principal component analysis (PCA) was applied to identify the most significant differences (marker bands) in SERS data among the analyzed cells and to perform quantitative analysis of SERS data. Based on a developed PCA algorithm, the studied cell types were classified with an accuracy of 95% in 2D PCA to 98% in 3D PCA. These results clearly indicate the diagnostic efficiency for the discrimination between cancer and normal cells. In our approach, we exploited the one-step technology that exceeds most of the multi-stage CTCs analysis methods used and enables simultaneous filtration, enrichment, and identification of the tumor cells from blood specimens.

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A splicing factor SF2/ASF is a natural substrate for the kinase activity of human topoisomerase I. This study demonstrates that SF2/ASF inhibits DNA cleavage by human topoisomerase I induced by the anti-cancer agent camptothecin. The inhibition is independent of the phosphorylation status of SF2/ASF. We show that the inhibition did not result from binding of SF2/ASF to DNA that would hinder interactions between topoisomerase I and DNA. Neither it was a consequence of a loss of sensitivity of the enzyme to camptothecin. We provide evidence pointing to reduced formation of the cleavable complex in the presence of SF2/ ASF as a primary reason for the inhibition. This effect of SF2/ASF is reflected by inhibition of DNA relaxation catalysed by topoisomerase I.Keywords: topoisomerase I; SF2/ASF; camptothecin.Eukaryotic topoisomerase I (topo I) catalyses DNA relaxation and plays a key role in DNA replication, transcription and recombination in the cell [1]. Topo I is also a target for several anti-cancer drugs derived from the cytotoxic plant alkaloid camptothecin [2]. A transient intermediate of the enzyme's catalytic cycle is the cleavable complex, consisting of the enzyme linked covalently to one strand of DNA. This complex is stabilized by camptothecin and can be detected as topo I-associated DNA single-strand breaks [3]. These breaks are usually called camptothecin-induced DNA cleavage. Camptothecin-induced DNA cleavage is essentially reduced by binding of SV40 T antigen [4] or ATP [5] to topo I. The complex is not detected for several mutated forms of the enzyme that are resistant to camptothecin (reviewed in [6]). Sensitivity to camptothecin is also diminished by dephosphorylation of topo I [7,8].Human topoisomerase I (htopo I) possesses a protein kinase activity which is specific towards serine residues of splicing factors containing a serine-arginine (SR) motif [9]. Phosphorylation of SR proteins is instrumental for the recruitment of these proteins to active sites of transcription in vivo [10] and for their activity as splicing factors [11]. SF2/ ASF is the main splicing factor containing an SR motif [12] phosphorylated by htopo I/kinase [9]. The binding site for SF2/ASF in htopo I is located between residues 135 and 175 [13]. This region is included in the N-terminal domain of the enzyme that in general is dispensable for both relaxation and binding of camptothecin [14,15]. However, detailed studies have recently revealed that the N-terminal domain influences the rate of relaxation and sensitivity of the enzyme to camptothecin [16].In the present work we report that the natural substrate for the kinase activity of htopo I, SF2/ASF protein, inhibits camptothecin-induced DNA cleavage by the enzyme. This effect results from reduced amount of the cleavable complex formed by htopo I in the presence of SF2/ASF. M A T E R I A L S A N D M E T H O D S Plasmids and strainsThe pRS426-based plasmid, containing complete human topo I cDNA under the control of the GAL1-10, was a gift from B. R. Knudsen (University of Aarhu...