Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme.

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

Effects of SRSF1 on subnuclear localization of topoisomerase I.

Subnuclear localization of topoisomerase I (top I) is determined by its DNA relaxation activity and a net of its interactions with in majority unidentified nucleolar and nucleoplasmic elements. Here, we recognized SR protein SRSF1 (Serine/arginine-rich splicing factor 1, previously known as SF2/ASF) as a new element of the net. In HeLa cells, overexpression of SRSF1 recruited top I to the nucleoplasm whereas its silencing concentrated it in the nucleolus. Effect of SRSF1 was independent of top I relaxation activity and was the best pronounced for the mutant inactive in relaxation reaction. In HCT116 cells where top I was not released from the nucleolus upon halting relaxation activity, it was also not relocated by elevated level of SRSF1. Out of remaining SR proteins, SRSF5, SRSF7, and SRSF9 did not influence the localization of top I in HeLa cells whereas overexpression of SRSF2, SRSF3, SRSF6, and partly SRSF4 concentrated top I in the nucleolus, most possibly due to the reduction of the SRSF1 accessibility. Specific effect of SRSF1 was exerted because of its distinct RS domain. Silencing of SRSF1 compensated the deletion of the top I N-terminal region, individually responsible for nucleoplasmic localization of the mutant, and restored the wild-type phenotype of deletion mutant localization. SRSF1 was essential for the camptothecin-induced clearance from the nucleolus. These results suggest a possible role of SRSF1 in establishing partition of top I between the nucleolus and the nucleoplasm in some cell types with distinct combinations of SR proteins levels.

Subnuclear Localization of Human Topoisomerase I.

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.

Controlled delivery of BID protein fused with TAT peptide sensitizes cancer cells to apoptosis.

BACKGROUND: Low 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. METHODS: Uptake 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. RESULTS: TAT-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. CONCLUSIONS: TAT-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.

Activities of topoisomerase I in its complex with SRSF1.

Human DNA topoisomerase I (topo I) catalyzes DNA relaxation and phosphorylates SRSF1. Whereas the structure of topo I complexed with DNA has been resolved, the structure of topo I in the complex with SRSF1 and structural determinants of topo I activities in this complex are not known. The main obstacle to resolving the structure is a contribution of unfolded domains of topo I and SRSF1 in formation of the complex. To overcome this difficulty, we employed a three-step strategy: identifying the interaction regions, modeling the complex, and validating the model with biochemical methods. The binding sites in both topo I and SRSF1 are localized in the structured regions as well as in the unfolded domains. One observes cooperation between the binding sites in topo I but not in SRSF1. Our results indicate two features of the unfolded RS domain of SRSF1 containing phosphorylated residues that are critical for the kinase activity of topo I: its spatial arrangement relative to topo I and the organization of its sequence. The efficiency of phosphorylation of SRSF1 depends on the length and flexibility of the spacer between the two RRM domains that uniquely determine an arrangement of the RS domain relative to topo I. The spacer also influences inhibition of DNA nicking, a prerequisite for DNA relaxation. To be phosphorylated, the RS domain has to include a short sequence recognized by topo I. A lack of this sequence in the mutants of SRSF1 or its spatial inaccessibility in SRSF9 makes them inadequate as topo I/kinase substrates.

Trimeric purine nucleoside phosphorylase: exploring postulated one-third-of-the-sites binding in the transition state.

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.

Overexpressed proteins may act as mops removing their ligands from the host cells: a case study of calf PNP.

Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes--moping of ligands from the host cells--may take place in the case of other proteins with high overexpression yield.

1.45 A resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state.

Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K(d) ( approximately 190pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position).

Native state dynamics and mechanical properties of human topoisomerase I within a structure-based coarse-grained model.

A coarse grained molecular dynamics model with an implicit solvent is used to elucidate properties of the human topoisomerase I. The model is defined through the native structure and it allows covering significantly longer time scales than in all atom simulations. Single residue and double residue motional characteristics are studied. The results are consistent with all atom simulations reported in the literature indicating usefulness of the model in further studies of this protein. Novel findings include broadening of the description of the dynamic behavior of the lip and hinge regions and a characterization of the motional properties of the RRM binding site of the enzyme. We also consider mechanical stretching of the protein and identify sources of the force peaks. The elastic properties of topoisomerase I are predicted to be average in comparison to other proteins, yielding a maximum force of resistance to pulling which should be of order 120 pN. The contact unraveling pattern is consistent with the understanding of the structure and function of the protein. We find supporting evidence for the hypothesis that the C-terminal domain acquires an ordered structure upon binding with the core enzyme even though it forms a molten globule when in isolation.

Fragment responsible for translocation in the N-terminal domain of human topoisomerase I.

The N-terminal domain is a fragment that binds proteins and anchors topoisomerase I in the nucleolus. As a separate polypeptide, it translocates from the nucleolus to nucleoplasm upon camptothecin treatment. In this paper, we show that the translocation depends on the short fragment of the domain (residues from 1 to 67). We also present a list of proteins that specifically bind to the fragment responsible for translocation.

RRM proteins interacting with the cap region of topoisomerase I.

RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54(nrb) and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1-topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the beta-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.

Overexpression, purification and characterization of functional calf purine nucleoside phosphorylase (PNP).

Calf PNP is a ubiquitous enzyme of the salvage metabolic pathway. The procedure for this enzyme production in large quantities is described. The coding sequence of bovine PNP was amplified from the calf spleen cDNA library and was inserted into an expression vector pET28a(+). The construct was transformed into Escherichia coli BL21(DE3) strain. The protein expression efficiencies in the presence and the absence of IPTG were compared. It was found that IPTG is not necessary for obtaining a large quantity of recombinant calf PNP: 35 mg from 1L cell culture. The enzyme was purified to 92% homogeneity by a two-step procedure consisting of gel filtration and ion exchange chromatography. The purity of recombinant enzyme is sufficient to form well diffracting single crystals. The basic kinetic parameters of recombinant PNP were determined and compared with the parameters of commercially available PNP from calf spleen. The specific activity in 50 mM phosphate buffer with inosine as a variable substrate (30.7 micromol min(-1)mg(-1)) and other kinetic parameters: Michaelis constants, maximal velocities, dissociation and inhibition constants, determined for several typical PNP ligands, are similar to the values published previously for non-recombinant calf spleen PNP. As expected for mammalian PNP, recombinant calf PNP was found to have no substrate activity vs adenosine. The overexpression and purification method of the recombinant calf PNP provides significant amounts of the enzyme, which can successfully replace the non-recombinant PNP.

Cloning, expression, purification, and some properties of calf purine nucleoside phosphorylase.

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.

Proteomic analysis of complexes formed by human topoisomerase I.

Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.

[Assessment of early complications in patients with abdominal aortic aneurysm after endovascular operations]. / Ocena wczesnych powiklan u chorych z tetniakiem aorty brzusznej leczonych endowaskularnie.

UNLABELLED: During past few years endovascular procedures at patients with abdominal aorta aneurysm (AAA) have widely replaced surgical treatment. The number of patients being operated by the use of this method has been growing. AIM OF THE STUDY: The aim of this study is to estimate early complications in patients with AAA treated by the use of bifurcated aortic stent-grafts. MATERIAL AND METHODS: Between the May 2002 and April 2003, 42 patients were qualified to endovascular operations (of the average age of 65). All of them represented a group of high operative risk patients. Patients were operated in epidural anesthesia (n=36) and in general intravenous anesthesia (n=6). In all the patients angiography was performed right after the operation to estimate the efficacy of treatment. The follow-up examinations proceeded after 1, 3, and 6 months. RESULTS: During the hospitalization, which lasted 7 days on average, clotting in one of the crura of the stent-graft in common iliac artery with the necessity of embolectomy, the some patient had to have an additional crossover by-pass implanted. There were also observed certain post-hospitalization complications which contained: a) clotting of stent-graft leg in one case, b) leak type I which demanded implantation of additional element of stent-graft in one case c) small leaks type I in 2 cases and type II in 2 cases which did not demand any further intervention, the type II leaks were observed in 3 cases 6 months after the operation, d) the increase of parietal circular thrombus inside the main part of the stent-graft in one case, e) complaints connected with the internal iliac artery occlusion were declared by one patient. CONCLUSIONS: The early results of AAA treatment by the use of bifurcated stent-grafts are satisfactory and connected with low rate of complications. Most of the early complications is connected with wrong qualification of patients, improper stent-graft choice and technical errors during the operation. Constant, periodical follow-up examinations (USG, angio CT) are necessary to estimate the late results.

[Endovascular treatment with bifurcated stent-grafts in patients with abdominal aortic aneurysms]. / Leczenie chorych z tetniakiem aorty brzusznej przy uzyciu rozwidlonych stent-graftów aortalnych.

UNLABELLED: During past few years endovascular procedures at patients with abdominal aorta aneurysm (AAA) have widely replaced surgical treatment. The interest put in endovascular treatment is high. The number of patients being operated by the use of this method has been growing. AIM OF THE STUDY: The objective of this article was the evaluation of the results of the endovascular treatment with 42 patients with abdominal aorta aneurysms. MATERIAL AND METHODS: All patients (38 men and 4 women, mean age 65 years) belong to the high risk group. The qualification for the endovascular treatment was after the clinical examination and angio-CT, abdominal and pelvis digital subtraction angiography (DSA). 36 operations were performed under supradural anesthesia and 6 under general anesthesia. Among all patients implanted bifurcated abdominal stent-grafts Zenith (COOK) fixing above the renal arteries. RESULTS: All operations were ended with success. Some early complications were. the leakage to sack of aneurysm, thrombosis of a leg of stent-graft, the curve of a leg, the growth of the thrombus in stent-graft. All patients had the control examinations (abdominal-rtg, pelvis-rtg, USG-color Doppler and angio-CT) immediately after the operation and successively after 1, 3 and 6 months. CONCLUSION: 1. The endovascular procedure among patients with AAA by means of bifurcated aortic stent-grafts is in many cases a principal method of treatment. 2. Early results of endovascular treatment are good. 3. Longer follow-up is needed for the evaluation of late results.

Synergy of BID with doxorubicin in the killing of cancer cells.

Overexpression of the BH3-interacting domain death agonist (BID) protein sensitizes certain cancer cell lines to apoptosis induced by anticancer agents, particularly by those acting through death receptors (e.g. TRAIL). Previously, we showed that recombinant BID fused with TAT cell penetrating peptide (TAT-BID) allowed for controlled delivery of BID to different cancer cell lines and moderately sensitized some of them to TRAIL or slightly to camptothecin. In the present study, we showed that TAT-BID delivered to HeLa cells strongly sensitized them to doxorubicin, as identified by cell viability and apoptosis assays. Another cell line sensitized to doxorubicin was PC3, whereas A549 and LNCaP cells were sensitized moderately or not at all, respectively. Sensitization was more pronounced at 1 µM doxorubicin administered for 48 h than for lower doses and shorter treatments. TAT-BID and doxorubicin may thus be considered as a potential therapeutic combination for cervical carcinoma and advanced prostate cancer treatment.

Lipid peroxidation product 4-hydroxy-2-nonenal modulates base excision repair in human cells.

Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway. One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H2O2 and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1,N(6)-ethenoadenine (ɛA) and 3,N(4)-ethenocytosine (ɛC), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re-ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H2O2 or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising ɛA and ɛC from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway.

Time schedule-dependent effect of the CK2 inhibitor TBB on PC-3 human prostate cancer cell viability.

Inhibitors of CK2 kinase inhibit cell proliferation and induce apoptosis in numerous cancer cell lines. Due to these properties, they are considered potentially useful in anticancer therapy. In this study, we show that the exact effect of the specific CK2 inhibitor TBB on PC-3 human prostate cancer cell viability depends on the time schedule of administration: it was not observed when the treatment was directly followed by the viability assay but it appeared when the treatment and the assay were separated by a 24-h incubation without the inhibitor. Such a pattern was maintained when the TBB treatment was combined with either camptothecin or TRAIL. The time schedule-dependence of cell viability was not reflected by a similar dependence of induction of apoptosis. Despite this, the schedule in which a treatment with the CK2 inhibitor precedes that with an anticancer drug seems to be a good choice for a potential therapy against androgen-refractory prostate cancer.

Poly(ADP-ribose) binds to the splicing factor ASF/SF2 and regulates its phosphorylation by DNA topoisomerase I.

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 SR-protein), 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.