Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription

The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a role in transcription and RNA processing. Yeast ESS1, a peptidyl-prolyl cis/trans isomerase, is involved in RNA processing and can associate with the CTD. Using several types of assays we could not find any evidence of an effect of Pin1, the human homolog of ESS1, on transcription by RNA polymerase II in vitro or on the expression of a reporter gene in vivo. However, an inhibitor of Pin1, 5-hydroxy-1,4-naphthoquinone (juglone), blocked transcription by RNA polymerase II. Unlike N-ethylmaleimide, which inhibited all phases of transcription by RNA polymerase II, juglone disrupted the formation of functional preinitiation complexes by modifying sulfhydryl groups but did not have any significant effect on either initiation or elongation. Both RNA polymerases I and III, but not T7 RNA polymerase, were inhibited by juglone. The primary target of juglone has not been unambiguously identified, although a site on the polymerase itself is suggested by inhibition of RNA polymerase II during factor-independent transcription of single-stranded DNA. Because of its unique inhibitory properties juglone should prove useful in studying transcription in vitro.

An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and γ irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5′ and 3′ ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2–20°C at 1 μM dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

Telomere dysfunction alters the chemotherapeutic profile of transformed cells

Telomerase inhibition has been touted as a novel cancer-selective therapeutic goal based on the observation of high telomerase levels in most cancers and the importance of telomere maintenance in long-term cellular growth and survival. Here, the impact of telomere dysfunction on chemotherapeutic responses was assessed in normal and neoplastic cells derived from telomerase RNA null (mTERC−/−) mice. Telomere dysfunction, rather than telomerase per se, was found to be the principal determinant governing chemosensitivity specifically to agents that induced double-stranded DNA breaks (DSB). Enhanced chemosensitivity in telomere dysfunctional cells was linked to therapy-induced fragmentation and multichromosomal fusions, whereas telomerase reconstitution restored genomic integrity and chemoresistance. Loss of p53 function muted the cytotoxic effects of DSB-inducing agents in cells with telomere dysfunction. Together, these results point to the combined use of DSB-inducing agents and telomere maintenance inhibition as an effective anticancer therapeutic approach particularly in cells with intact p53-dependent checkpoint responses.

Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.

Activation of the JC virus Tat-responsive transcriptional control element by association of the Tat protein of human immunodeficiency virus 1 with cellular protein Purα

JC virus is activated to replicate in glial cells of many AIDS patients with neurological disorders. In human glial cells, the human immunodeficiency virus 1 (HIV-1) Tat protein activates the major late promoter of JC virus through a Tat-responsive DNA element, termed upTAR, which is a recognition site for cellular Purα, a sequence-specific single-stranded DNA binding protein implicated in cell cycle control of DNA replication and transcription. Tat interacts with two leucine-rich repeats in Purα to form a complex that can be immunoprecipitated from cell extracts. Tat enhances the ability of purified glutathione S-transferase-Purα (GST-Purα) to bind the upTAR element. Tat acts synergistically with Purα, in a cell-cycle-dependent manner, to activate transcription at an upTAR element placed upstream of a heterologous promoter. Since Purα is ubiquitously expressed in human cells and since PUR elements are located near many promoters and origins of replication, the Tat-Purα interaction may be implicated in effects of HIV-1 throughout the full range of HIV-1-infected cells.

Communication between noncontacting macromolecules

We present a quantitative experimental demonstration of solvent-mediated communication between noncontacting biopolymers. We show that changes in the activity of a solvent component brought about by a conformational change in one biopolymer can result in changes in the physical properties of a second noncontacting biopolymer present in solution. Specifically, we show that the release of protons on denaturation of a donor polymer (in this case, a four-stranded DNA tetraplex, iDNA) modulates the melting temperature of a noncontacting, acceptor polymer [in this case poly(A)]. In addition to such proton-mediated cross talk, we also demonstrate counterion-mediated cross talk between noncontacting biopolymers. Specifically, we show that counterion association/release on denaturation of native salmon sperm DNA (the donor polymer) can modulate the melting temperature of poly(dA)⋅poly(dT) (the acceptor polymer). Taken together, these two examples demonstrate how poly(A) and poly(dA)⋅poly(dT) can serve as molecular probes that report the pH and free salt concentrations in solution, respectively. Further, we demonstrate how such through-solvent dialogue between biopolymers that do not directly interact can be used to evaluate (in a model-free manner) association/dissociation reactions of solvent components (e.g., protons, sodium cations) with one of the two biopolymers. We propose that such through-solution dialogue is a general property of all biopolymers. As a result, such solvent-mediated cross talk should be considered when assessing reactions of multicomponent systems such as those that exist in essentially all biological processes.

The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication.

We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication. Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2). The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE. Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication. Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA. The interaction with duplex DNA occurs at the termini. N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen. Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication.

Molecular mechanics calculations of the riboacetal internucleotide linkage in double and triple helices.

Structures of Watson-Crick base paired 15-nucleobase oligomer strands in A-type or B-type conformation in which one strand [a strand of alternating nucleotide and riboacetal thymidine nucleoside (RT) units, RP] is DNA and the other is composed of alternating nucleotides and riboacetal nucleosides have been studied by molecular mechanics. Analogously, oligomer strands of RNA in place of DNA have been modeled. The calculations indicate that the RP strand is more stable when complexed in an A-type duplex relative to a B-type form and that this conformational preference is presumably due to the more uniform nature of the former. Nearly planar ribose rings were more commonly observed in the minimized structures of the B-type DNA.RP duplexes as compared with A-type duplexes, despite the fact that planar ribofuranose rings are known to be energetically unfavorable in oligonucleotides. Computed relative stabilities of all duplexes containing the RP strand suggest that such heteroduplexes are less stable than the corresponding double-stranded DNA and double-stranded RNA species. These findings are in agreement with experimental results which show, when equivalent sequences were compared, that a DNA.RNA control forms a more stable duplex than RP hound to a complementary single-stranded RNA strand. In contrast, molecular mechanics studies of complementary triple-helical (DNA)2.RP, (DNA)2.DNA, and (DNA)2.RNA structures indicate that the binding of RP as a Hoogsteen strand stabilizes the underlying duplex to a greater extent compared with native oligonucleotides. These calculations suggest that puckering of the ribose ring in the riboacetal linkage leads to a more favorable interaction with a complementary nucleic acid target than the proposed planar geometry and that this puckering may account for the enhanced binding of RP to a double-stranded target.

Presynaptic association of Rad51 protein with selected sites in meiotic chromatin.

Eukaryotic homologs of Escherichia coli Rec-A protein have been shown to form nucleoprotein filaments with single-stranded DNA that recognize homologous sequences in duplex DNA. Several recent reports in four widely diverse species have demonstrated the association of RecA homologs with meiotic prophase chromatin. The current immunocytological study on mouse spermatocytes and oocytes shows that a eukaryotic homolog, Rad5l, associates with a subset of chromatin sites as early as premeiotic S phase, hours before either the appearance of precursors of synaptonemal complexes or the initiation of synapsis. When homologous chromosomes do begin to pair, the Rad5l-associated sequences are sites of initial contact between homologues and of localized DNA synthesis. Distribution of Rad5l foci on the chromatin of fully synapsed bivalents at early pachynema corresponds to an R-band pattern of mitotic chromosomes. R-bands are known to be preferred sites of both synaptic initiation and recombination. The time course of appearance of Rad51 association with chromatin, its distribution, and its interaction with other Rad5l-associated sequences suggests that it plays an important role preselection of sequences and synaptic initiation.

Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein.

Type II restriction endonucleases are dimers of two identical subunits that together form one binding site for the double-stranded DNA substrate. Cleavage within the palindromic recognition site occurs in the two strands of the duplex in a concerted manner, due to the action of two catalytic centers, one per subunit. To investigate how the two identical subunits of the restriction endonuclease EcoRV cooperate in binding and cleaving their substrate, heterodimeric versions of EcoRV with different amino acid substitutions in the two subunits were constructed. For this purpose, the ecorV gene was fused to the coding region for the glutathione-binding domain of the glutathione S-transferase and a His6-tag, respectively. Upon cotransformation of Escherichia coli cells with both gene fusions stable homo- and heterodimers of the EcoRV variants are produced, which can be separated and purified to homogeneity by affinity chromatography over Ni-nitrilotriacetic acid and glutathione columns. A steady-state kinetic analysis shows that the activity of a heterodimeric variant with one inactive catalytic center is decreased by 2-fold, demonstrating that the two catalytic centers operate independently from each other. In contrast, heterodimeric variants with a defect in one DNA-binding site have a 30- to 50-fold lower activity, indicating that the two subunits of EcoRV cooperate in the recognition of the palindromic DNA sequence. By combining a subunit with an inactive catalytic center with a subunit with a defect in the DNA-binding site, EcoRV heterodimers were produced that only nick DNA specifically within the EcoRV recognition sequence.

Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

Pathogenic autoantibodies are routinely generated during the response to foreign antigen: a paradigm for autoimmune disease.

The immune system's ability to distinguish self and nonself is essential for both host defense against foreign agents and protection of self-antigens from autoimmune destruction. Such discrimination is complicated by extensive structural homology shared between foreign and self antigens. One hypothesis to explain the development of an autoimmune response is that some B cells activated by foreign antigen acquire, through somatic mutation, specificity for both the eliciting foreign antigen and self antigen. If such clones arise frequently, there must be a mechanism for their elimination. We have analyzed the extent of autoreactivity arising in a nonautoimmune host during the response to a foreign antigen. To overcome the process of apoptosis in primary B cells that might routinely eliminate autoreactive clones, we generated B-cell hybridomas from spleen cells of immunized mice by using a fusion partner constitutively expressing bcl-2. Multiple lines were obtained that recognize simultaneously the hapten phosphorylcholine and the self antigen double-stranded DNA. This dual specificity was not present early but was detected by day 10 after immunization. Some of these cross-reactive antibodies deposit in kidneys in a pattern similar to what is seen in autoimmune disease. These results demonstrate that autoantibodies arise at a high frequency as part of a response to foreign antigen. It has previously been shown that autoreactivity is regulated by central deletion; these data demonstrate a need for negative selection in peripheral lymphoid organs also, to regulate autoantibodies acquiring their self-specificity by somatic mutation.

Anti-nuclear antibody production and immune-complex glomerulonephritis in BALB/c mice treated with pristane.

The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.

Oligonucleotide N3'-->P5' phosphoramidates.

Synthetic oligonucleotides and their analogs have attracted considerable interest recently. These compounds may lead to highly specific therapeutic agents, as well as to powerful diagnostic tools. Here, we present the synthesis of uniformly modified oligodeoxyribonucleotide N3'-->P5' phosphoramidates containing 3'-NHP(O)(O-)O-5' internucleoside linkages and the study of their hybridization properties. Thermal dissociation experiments show that these compounds form very stable duplexes with single-stranded DNA, RNA, and with themselves following Watson-Crick base pairing. The duplex thermal stability was enhanced by 2.2-2.6 degrees C per modified linkage compared with phosphodiesters. The structure of complexes formed by phosphoramidates closely resembles that of RNA oligomers and corresponds to an A form, as judged by CD spectroscopy. N3'-->P5' phosphoramidates also form stable triplexes with double-stranded DNA under near-physiological conditions when natural phosphodiesters fail to do so. Physicochemical characteristics of the amidates are similar to those of RNA oligomers, even though they are composed of 2'-deoxyfuranose-based nucleosides.