Sequence-specific Single-stranded DNA Research Articles

The crystal structure of the human smacovirus 1 Rep domain.

Replication initiator proteins (Reps) from the HUH endonuclease family process specific single-stranded DNA sequences to initiate rolling-circle replication in viruses. Here, the first crystal structure of the apo state of a Rep domain from the smacovirus family is reported. The structure of the human smacovirus 1 Rep domain was obtained at 1.33 Å resolution and represents an expansion of the HUH endonuclease superfamily, allowing greater diversity in bioconjugation-tag applications.

Detection of Pancreatic Cancer miRNA with Biocompatible Nitrogen-Doped Graphene Quantum Dots.

Early-stage pancreatic cancer remains challenging to detect, leading to a poor five-year patient survival rate. This obstacle necessitates the development of early detection approaches based on novel technologies and materials. In this work, the presence of a specific pancreatic cancer-derived miRNA (pre-miR-132) is detected using the fluorescence properties of biocompatible nitrogen-doped graphene quantum dots (NGQDs) synthesized using a bottom-up approach from a single glucosamine precursor. The sensor platform is comprised of slightly positively charged (1.14 0.36 mV) NGQDs bound via stacking and/or electrostatic interactions to the negatively charged (−22.4 6.00 mV) bait ssDNA; together, they form a complex with a 20 nm average size. The NGQDs’ fluorescence distinguishes specific single-stranded DNA sequences due to bait–target complementarity, discriminating them from random control sequences with sensitivity in the micromolar range. Furthermore, this targetability can also detect the stem and loop portions of pre-miR-132, adding to the practicality of the biosensor. This non-invasive approach allows cancer-specific miRNA detection to facilitate early diagnosis of various forms of cancer.

A novel cloth-based supersandwich electrochemical aptasensor for direct, sensitive detection of pathogens

Traditional detection methods for food-borne pathogens are usually expensive and laborious, so there is an urgent need for an economical, facile and sensitive method. In this work, a novel cloth-based supersandwich electrochemical aptasensor (CSEA) is firstly developed for direct detection of pathogens. Carbon ink- and wax-based screen-printing is used to make cloth-based electrodes and hydrophilic/hydrophobic regions respectively to fabricate the sensing devices. Two well-designed, specific single-stranded DNA sequences arise a cascade hybridization reaction to form the DNA supersandwich (DSS) whose grooves can be inserted by methylene blue (MB), which effectively amplifies the current signal to greatly improve the detection sensitivity. Taking the detection of Salmonella typhimurium (S. typhimurium) as an example, the aptamers bind to S. typhimurium to form the target-aptamers complex, which can simultaneously bind to the capture probe and DSS, resulting in detection of S. typhimurium. Moreover, the addition of tail sequences of aptamer makes the proposed CSEA versatile. Under optimized conditions, the electrochemical signal increases linearly with the logarithm of S. typhimurium concentration over the range from 102 to 108 CFU mL−1, with a limit of detection of 16 CFU mL−1. Additionally, the CSEA efficiently determined the levels of S. typhimurium in milk samples. Experimental results illustrate that the fabricated CSEA is sensitive, specific, reproducible and stable. Moreover, when Ru(bpy)32+ replaces MB, the electrochemiluminescence (ECL) can be performed. Thus, for the proposed sensing strategy, the dual-mode detection of electrochemistry and ECL is easily realized.

Chirality dependent corona phase molecular recognition of DNA-wrapped carbon nanotubes

Corona phase molecular recognition (CoPhMoRe) is a phenomenon whereby a polymer or surfactant corona phase wrapped around a nanoparticle selectively recognizes a particular molecule. The method can potentially generate non-biological, synthetic molecular recognition sites, analogous to antibodies, for a broad range of biomedical applications, including new types of sensors, laboratory and clinical assays, as well as inhibitors and targeted therapeutics. In this work, we utilize near infrared fluorescent single-walled carbon nanotubes (SWNTs) wrapped with specific single stranded DNA sequences to explore the (n,m) chirality dependence of CoPhMoRe. Specific DNA oligonucleotide sequences are known to recognize and interact uniquely with certain (n,m) SWNTs enabling their enrichment in ion exchange chromatography. We explore the CoPhMoRe effect using corona phases constructed from a library of 24 such sequences, screening against a biomolecule panel that includes common neurotransmitters, amino acids, saccharides and riboflavin. Example sequences include (ATT)4, (TAT)4 and (ATTT)3 which recognize (7,5), (6,5) and (8,4) SWNTs, respectively. We find that these recognition sequences indeed form CoPhMoRe phases that are distinct among SWNT chiralities, and appear to pack more densely as to exclude analyte adsorption on the chirality they recognize. These results have encouraging implications for the controlled design of CoPhMoRe phases for biomedical applications.

A label-free biosensor for DNA detection based on ligand-responsive G-quadruplex formation

A facile and label-free assay with label-free molecular beacons (MBs) and fluorescent dye N-methyl mesoporphyrin IX (NMM) was developed for the detection of specific single-stranded DNA sequences. It was demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 20 bases) of RNA fragment of human immunodeficiency virus (HIV) as model systems. In the absence of target DNA, the MBs were in the stem-closed form, the G-quadruplex structure could not form and the fluorescence signal of NMM was very low. In the presence of target DNA the MBs turned “Off” to “On”, thus promoting the formation of G-quadruplex which could greatly enhance the fluorescence of NMM. This biosensor was simple in design, fast in operation, and more convenient and promising than other methods. It took less than 30min to finish and its detection limit was 1.4nM. No sophisticated experimental techniques or chemical modification for DNA sequences were required. This new approach could be widely applied to sensitive and selective nucleic acids detection.

A label-free signal amplification assay for DNA detection based on exonuclease III and nucleic acid dye SYBR Green I

We have developed a new fluorescence method for specific single-stranded DNA sequences with exonuclease III (Exo III) and nucleic acid dye SYBR Green I. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 27 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of the target DNA, the hairpin-probe is in the stem-closed structure, the fluorescence of SYBR Green I is very strong. In the presence of the target DNA, the hairpin-probe hybridizes with the target DNA to form double-stranded structure with a blunt 3′-terminus. Thus, in the presence of Exo III, only the 3′-terminus of probe is subjected to digestion. Exo III catalyzes the stepwise removal of mononucleotides from this terminus, releasing the target DNA. The released target DNA then hybridizes with another probe, whence the cycle starts anew. The signal of SYBR Green I decreases greatly. This system provides a detection limit of 160pM, which is comparable to the existing signal amplification methods that utilized Exo III as a signal amplification nuclease. Due to the unique property of Exo III, this method shows excellent detection selectivity for single-base discrimination. More importantly, superiors to other methods based on Exo III, these probes have the advantages of easier to design, synthesize, purify and thus are much cheaper and more applicable. This new approach could be widely applied to sensitive and selective nucleic acids detection.

DNA Scanning Mechanism of a Translocating Motor Protein

Unrepaired DNA breaks can lead to genomic instability or cell death. For repair by the ubiquitous homologous recombination pathway, broken ends are first processed to produce a 3'-ssDNA overhang. In Bacillus subtilis, this reaction is catalysed by AddAB helicase-nuclease complexes; motor proteins that unwind the DNA duplex and degrade the nascent single-strands in a manner regulated by specific single-stranded DNA sequences called Chi recombination hotspots (Yeeles and Dillingham, 2007; Yeeles et al., 2011). We have used Magnetic Tweezers to investigate the real-time dynamics of AddAB translocation on dsDNA and the effect of recombination hotspot recognition on this process. AddAB translocation traces showed a complex appearance with variable velocities between 200-400 bp/s at room temperature. We found that AddAB was prone to stochastic pausing in areas which contained many Chi-like sequences. Experiments using an AddAB mutant that is unable to recognize Chi strongly suggest that this pausing is due to transient recognition of Chi-like sequences and highlight the antagonistic nature of DNA translocation and sequence specific DNA recognition activities. Experiments using substrates containing bona fide Chi sequences showed that AddAB also pauses at Chi, but these events are longer and not exponentially distributed, suggesting a multistep process. We propose a model for the recognition of Chi and Chi-like sequences to explain the origins of this pausing behavior during failed or successful hotspot recognition.Yeeles, J. T., and Dillingham, M. S. (2007). A dual-nuclease mechanism for DNA break processing by AddAB-type helicase-nucleases. J Mol Biol 371, 66-78.Yeeles, J. T., van Aelst, K., Dillingham, M. S., and Moreno-Herrero, F. (2011). Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease. Molecular Cell 42, 806-816.

TLS/FUS (translocated in liposarcoma/fused in sarcoma) regulates target gene transcription via single-stranded DNA response elements

TLS/FUS (TLS) is a multifunctional protein implicated in a wide range of cellular processes, including transcription and mRNA processing, as well as in both cancer and neurological disease. However, little is currently known about TLS target genes and how they are recognized. Here, we used ChIP and promoter microarrays to identify genes potentially regulated by TLS. Among these genes, we detected a number that correlate with previously known functions of TLS, and confirmed TLS occupancy at several of them by ChIP. We also detected changes in mRNA levels of these target genes in cells where TLS levels were altered, indicative of both activation and repression. Next, we used data from the microarray and computational methods to determine whether specific sequences were enriched in DNA fragments bound by TLS. This analysis suggested the existence of TLS response elements, and we show that purified TLS indeed binds these sequences with specificity in vitro. Remarkably, however, TLS binds only single-strand versions of the sequences. Taken together, our results indicate that TLS regulates expression of specific target genes, likely via recognition of specific single-stranded DNA sequences located within their promoter regions.

Hybridization-triggered isothermal signal amplification coupled with MutS for label-free and sensitive fluorescent assay of SNPs

Combining the specific recognization of MutS protein for mismatched DNA sequences with the target-driven molecular switch that acts as both the template and primer of the polymerization reaction, a new label-free and sensitive fluorescent assay strategy for specific single-stranded DNA sequences or SNPs is proposed.

Reduced working electrode based on fullerene C60 nanotubes@DNA: Characterization and application

Fullerene C60 nanotubes (FNTs) were functionalized with sequence-specific single-stranded DNA to form a kind of complexes (FNT@DNA), which could be brought efficiently into aqueous solution. The dispersed FNT@DNA could form a layer of stable film on the surface of glassy carbon electrode (GCE). In the Britton–Robinson buffer solution of pH ≥7.0, the FNT@DNA modified on the GCE presented an irreversible two-step six-electron transfer reduction reaction. The reduced modified electrode had a rather wide electrochemical window and could be used as a functionalized working electrode, which showed a good enrichment capability towards the positively charged molecules. The selective detection of dopamine in the presence of a high amount of ascorbic acid could be realized at the reduced FNT@DNA-modified GCE in neutral buffer solution.

Structural and functional characterization of the core single‐stranded DNA‐binding domain of purine‐rich element binding protein B (Purβ)

Purβ is a sequence‐specific single‐stranded DNA (ssDNA)‐binding protein that represses smooth muscle α‐actin gene transcription in stress‐activated fibroblasts and smooth muscle cells via cooperative interaction with two sites in the purine‐rich strand of a 5′ enhancer element (dubbed PE32‐F). To better understand how the modular structure of Purβ dictates its specific ssDNA‐binding function, we evaluated the interaction of purified recombinant Purβ with PE32‐F using spectroscopic and biochemical approaches. Results of intrinsic protein fluorescence and circular dichroism analyses pointed to a ssDNA‐induced change in protein conformation. Consistent with this interpretation, Purβ was protected from proteolysis when complexed with PE32‐F. Limited tryptic digestion of Purβ liberated a core ~30 kDa fragment spanning residues 29‐305 as determined by mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to dimerize. Quantitative ssDNA‐binding assays revealed that the core domain binds to PE32‐F with similar specificity but increased affinity compared to full‐length Purβ and a truncated 37‐263 mutant absent a putative amphipathic helix‐forming module. These findings provide new insight into the structural basis for the specific and high affinity binding of Purβ to ssDNA. NIH R01 HL054281, T32 HL007594, P20 RR16462.

Uncovering pathways in DNA oligonucleotide hybridization via transition state analysis.

DNA hybridization plays a central role in biology and, increasingly, in materials science. Yet, there is no precedent for examining the pathways by which specific single-stranded DNA sequences interact to assemble into a double helix. A detailed model of DNA is adopted in this work to examine such pathways and to determine the role of sequence, if any, on DNA hybridization. Transition path sampling simulations reveal that DNA rehybridization is prompted by a distinct nucleation event involving molecular sites with approximately four bases pairing with partners slightly offset from those involved in ideal duplexation. Nucleation is promoted in regions with repetitive base pair sequence motifs, which yield multiple possibilities for finding complementary base partners. Repetitive sequences follow a nonspecific pathway to renaturation consistent with a molecular "slithering" mechanism, whereas random sequences favor a restrictive pathway involving the formation of key base pairs before renaturation fully ensues.

Lack of SSBP2, a Candidate Leukemia Suppressor, Promotes T Cell Lymphomagenesis in p53 Null Mice.

Our laboratory positionally cloned a candidate myeloid leukemia suppressor gene, sequence-specific single-stranded DNA binding protein 2 (SSBP2) from chromosome 5q13.3-5q14.1 interval. SSBP2 expression is lost in primary acute myelogenous leukemia (AML) and myelodysplasia (MDS) cells. AML cells induced to express SSBP2 show growth arrest and partial differentiation. SSBP2 directly binds the adaptor protein Ldb1 (LIM domain-binding protein1), which in turn binds LIM domain containing nuclear factors. Although LDB1, SSBP2 containing transcriptional complexes are ubiquitously expressed, they have been best characterized in erythroid differentiation. Stabilization of Ldb1 by its direct interaction with SSBP2 upregulated the transcriptional activity of the Ldb1-Lmo2-E2A/TAL1-containing complex on an erythroid specific gene promoter. Here, we report the generation and characterization of Ssbp2 null mice. Mice with homozygous disruption of Ssbp2 (Ssbp2−/−) are viable and fertile, with no gross hematopoietic abnormalities observed up to six months of age. In order to examine if and how loss of SSBP2 promotes transformation, we crossed Ssbp2−/− mice with p53−/− null mice. Ssbp2−/−, p53−/− mice succumbed to thymic lymphomas with a median survival of 122 days compared to a median survival of 182 days for p53−/− mice (P = 0.0002). Moreover, the spectrum of tumors observed in the Ssbp2−/−, p53−/− mice included 62.5% thymic lymphomas (20/32), whereas only 26.7% of the p53−/− mice developed thymic lymphomas (8/30). In addition, Ldb1 protein levels but not transcript levels were decreased in the thymi of Ssbp2−/−, and Ssbp2−/−, p53−/− mice. Thus, loss of Ssbp2 appears to abrogate Ldb1-Lmo2-mediated T cell differentiation by promoting Ldb1 degradation. Such a block in differentiation cooperates with the loss of cell cycle arrest and apoptosis due to loss of p53. Our findings demonstrate for the first time a functional cooperation between the candidate leukemia suppressor SSBP2 and tumor suppressor p53. Furthermore, these results suggest the Ldb1-Lmo2 complexes to be novel targets for therapeutic intervention in hematologic malignancies.

Expression and Prognostic Impact of Candidate Suppressor Sequence-Specific Single-Stranded DNA Binding Protein (SSBP2) in AML Using Reverse Phase Proteins Arrays (RPPA).

Sequence-specific single-stranded DNA binding protein 2 (SSBP2) is a candidate tumor suppressor gene, located at chromosome 5q13.3, that is thought to regulate hematopoietic growth and differentiation. Loss of SSPB2 expression is associated with deletions of chromosome 5 and replacement in cell lines induces partial differentiation and loss of clonogencity. Using a highly specific antibody, we studied the level of expression and prognostic relevance of SSBP2 protein expression (along with 51 other total and phospho proteins as described in ASH abstract #107, 2006) in leukemia-enriched samples from 423 patients (258 newly diagnosed, 47 primary refractory, 118 relapse 1, 2 or refractory) using high-throughput reverse phase protein array technology (RPPA). Levels of SSBP2 were lower, equal and higher than that of normal CD34+ cells in 2.7%, 58.7% and 11.6% of cases respectively. All 7 cases with low levels were female and 21/30 with high levels were male. The expression of SSBP2 was not equally distributed in the different FAB subtypes with the lowest median levels in M5 and the highest in M0, M1 and M2. Levels were significantly higher in favorable cytogenetics (t(8;21) and Inv (16) compared to intermediate (p=0.05) or unfavorable (p=0.008) cytogenetics. Median levels were lowest in cases with cytogenetic abnormalities on chromosome 5 or with an 11q23 abnormality. Levels were significantly higher in primary refractory and relapse samples compared to diagnostic samples. The levels of SSBP2 expression was strongly positively correlated with expression levels of anti-apoptotic proteins BCL2, Bcl-XL, caspase inhibitors SMAC, Survivin and XIAP, and inversely correlated with inactivated Bad-p112. Expression also was positively correlated with proliferation regulating proteins β-Catenin, Cyclin D, GSK3, Myc, P27, as well as VEGF receptor neuropilin and P53. Levels were strongly negatively correlated with activated signal transduction proteins (STP) including phosphorylated AKT (phosphorylated on threonine 308 or serine 473), pMEK, pERK2, pP38, pPKCα and as well as activated P70S6K. For outcome analysis martingale residuals were utilized to define an optimal cutpoint for dichotomization into groups with higher (n=130, 111 treated) vs. lower (n=128, 106 treated) SSBP2. Complete remission rates (58% vs. 63%, p=0.39), relapse rates (53% vs. 49%, P=0.59, and median remission duration (37 vs. 39 weeks, p=0.37) did not differ between patients with lower vs. higher SSBP2. However patients with higher SSPB2 levels had significantly longer median survival (40 vs. 31 weeks, p=0.015) compared to patients with lower SSBP2 levels. In summary, SSBP2 expression is heterogeneous in AML ranging from low in the poor and mixed prognosis subsets and high in the good prognosis t(8;21). Consistent with its demonstrated role in hematopoietic differentiation, the levels correlate negatively with expression of proliferation enhancing and STP. Nonetheless, the significance of positive correlation with antiapoptotic proteins is unclear at present. But the association with favorable longer overall survival suggests that therapeutic agents targeting the SSBP2 pathway function might have efficacy in AML.

Roles of Active Site Residues and the HUH Motif of the F Plasmid TraI Relaxase

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.

Crosstalk between Primase Subunits Can Act to Regulate Primer Synthesis in trans

The coordination of primase function within the replisome is an essential but poorly understood feature of lagging strand synthesis. By using crystallography and small-angle X-ray scattering (SAXS), we show that functional elements of bacterial primase transition between two dominant conformations: an extended form that uncouples a regulatory domain from its associated RNA polymerase core and a compact state that sequesters the regulatory region from the site of primer synthesis. FRET studies and priming assays reveal that the regulatory domain of one primase subunit productively associates with nucleic acid that is bound to the polymerase domain of a second protomer in trans. This intersubunit interaction allows primase to select initiation sites on template DNA and implicates the regulatory domain as a "molecular brake" that restricts primer length. Our data suggest that the replisome may cooperatively use multiple primases and this conformational switch to control initiation frequency, processivity, and ultimately, Okazaki fragment synthesis.

SSBP2, a candidate tumor suppressor gene, induces growth arrest and differentiation of myeloid leukemia cells

Acute myelogenous leukemia (AML) is the most common leukemia in adults with clonal proliferation of myeloid stem cells. Two or more cooperating mechanisms, namely block in differentiation, enhanced proliferation and resistance to programmed cell death, underlie this neoplastic transformation. Nonrandom, complete and partial deletions of chromosome 5 are common anomalies in AML. Using positional cloning strategies, we characterized an evolutionarily conserved candidate myeloid leukemia suppressor gene encoding sequence-specific single-stranded DNA binding protein 2 (SSBP2) from chromosome 5q13.3, a locus that is frequently deleted in AML. Recent studies in Drosophila and Xenopus demonstrate a pivotal role for SSBPs in embryonic differentiation. In mammals, SSBP2 is one of three highly related and ubiquitously expressed genes. Here, we identify frequent loss of SSBP2 protein expression in human AML cell lines using highly specific antibodies. Furthermore, inducible expression of SSBP2 in the AML cell line U937 leads to loss of clonogenicity, G1 arrest and partial differentiation. Remarkably, inducible expression of SSBP2 is accompanied by downregulation of C-MYC expression. Our findings are consistent with human SSBP2 being a novel regulator of hematopoietic growth and differentiation, whose loss confers a block in differentiation advantage to myeloid leukemic cells.

Trax (Translin-associated Factor X), a Primarily Cytoplasmic Protein, Inhibits the Binding of TB-RBP (Translin) to RNA

Trax (Translin-associated factor X) has been shown to interact with TB-RBP/Translin by its coimmunoprecipitation and in yeast two-hybrid assays. Here we demonstrate that Trax is widely expressed, does not bind to DNA or RNA, but forms heterodimers with TB-RBP under reducing conditions. The heterodimer of TB-RBP and Trax inhibits TB-RBP binding to RNA, but enhances TB-RBP binding to specific single stranded DNA sequences. The in vitro interactions between TB-RBP and Trax are confirmed by similar interactions in the yeast two-hybrid system. Cell fractionation and confocal microscope studies reveal that Trax is predominantly cytoplasmic. In contrast, TB-RBP is present in both the nuclei and cytoplasm of transfected cells and uses a highly conserved nuclear export signal to exit nuclei. In addition to a leucine zipper, two basic domains in TB-RBP are essential for RNA binding, but only one of these domains is needed for DNA binding. Trax restores DNA binding to TB-RBP containing an altered form of this domain. These data suggest that Trax-TB.RBP interactions modulate the DNA- and RNA-binding activity of TB-RBP.

Ras-induced colony formation and anchorage-independent growth inhibited by elevated expression of Puralpha in NIH3T3 cells.

Levels of Puralpha, a conserved, sequence-specific single-stranded DNA and RNA binding protein, fluctuate during the cell cycle, declining at the onset of S-phase and peaking at mitosis. In early G1 Puralpha is associated with the hypophosphorylated form of the retinoblastoma protein, Rb. Microinjection of purified Puralpha into NIH3T3 cells arrests the cell cycle at either G1/S or G2/M checkpoints with distinct morphological consequences. Here we ask whether expression of Puralpha can affect colony formation and anchorage-independent growth in ras-transformed NIH3T3 cells. Two to five-fold elevated levels of Puralpha in stably-transfected cell lines retard entry into and progression through S phase in both ras-transformed and non-transformed cells. Puralpha significantly inhibits colony formation by ras-transformed cells but not by non-transformed cells. In addition, cells transfected to express Puralpha formed only about 1/5 the number of large colonies in soft agar as control-transfected cells, demonstrating a marked inhibition of anchorage-independent growth by Puralpha. Biochemical analysis of nuclear and cytoplasmic Puralpha proteins and confocal microscopic analysis of Puralpha location indicate that access of Puralpha to the nucleus is controlled by both protein modification and sequence domains within the protein. Analyses of deletion mutants identify Puralpha domains mediating nuclear exclusion, including several potential destruction motifs and a PEST sequence at aa's 215-231. In the nucleus Puralpha colocalizes with CDK2 and cyclin A. Puralpha and cyclin D1, however, do not colocalize in the nucleus. At mitosis Puralpha is visualized about the condensed chromosomes and in the cytoplasm, where it colocalizes with cyclin B1. The data indicate that the ability of Puralpha to interact with proteins regulating cell proliferation and transformation is controlled by signals that govern its intracellular localization.

Association of Pur alpha and E2F-1 suppresses transcriptional activity of E2F-1.

Protein-protein interaction can play an important role in the control of several biological events including gene transcription, replication and cell proliferation. E2F-1 is a DNA-binding transcription factor which, upon interaction with its target DNA sequence, induces expression of several S phase specific genes allowing progression of the cell cycle. Evidently, the activity of this protein is modulated by its cellular partner, pRb, which in the hypophosphorylated form, binds to E2F-1 and inactivates its transcriptional ability. In this study, we have demonstrated that expression of a sequence-specific single-stranded DNA binding protein, Pur alpha, in cells decreases the ability of E2F-1 to exert its transcriptional activity upon the responsive promoter derived from DHFR. Results from band shift experiments revealed that while Pur alpha does not recognize the double-stranded DNA fragment containing the E2F-1 binding site, it has the ability to inhibit E2F-1 interaction with its target DNA sequence. Results from GST pull-down assays and the combined immunoprecipitation/Western blot analysis of nuclear extracts revealed a direct association of E2F-1 with Pur alpha in the absence of the DNA molecule containing the E2F-1 binding site. The association of Pur alpha with E2F-1 may increase the stability of E2F-1, as a higher level of E2F-1 was detected in cells coexpressing Pur alpha and E2F-1. The importance of these observations with respect to the role of Pur alpha in the control of cell cycle progression is discussed.