Short DNA Fragments Are a Hallmark of Heavy Charged-Particle Irradiation and May Underlie Their Greater Therapeutic Efficacy.

Growing interest in proton and heavy ion therapy has reinvigorated research into the fundamental biological mechanisms underlying the therapeutic efficacy of charged-particle radiation. To improve our understanding of the greater biological effectiveness of high-LET radiations, we have investigated DNA double-strand breaks (DSBs) following exposure of plasmid DNA to low-LET Co-60 gamma photon and electron irradiation and to high-LET Beryllium and Argon ions with atomic force microscopy. The sizes of DNA fragments following radiation exposure were individually measured to construct fragment size distributions from which the DSB per DNA molecule and DSB spatial distributions were derived. We report that heavy charged particles induce a significantly larger proportion of short DNA fragments in irradiated DNA molecules, reflecting densely and clustered damage patterns of high-LET energy depositions. We attribute the enhanced short DNA fragmentation following high-LET radiations as an important determinant of the observed, enhanced biological effectiveness of high-LET irradiations.

RNA helicase A activity is inhibited by oncogenic transcription factor EWS-FLI1.

RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy.

Hepatitis delta antigen requires a flexible quasi-double-stranded RNA structure to bind and condense hepatitis delta virus RNA in a ribonucleoprotein complex.

UNLABELLED: The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2'OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg. IMPORTANCE: RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex.

STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain.

Activation of STAT3 in cancers leads to gene expression promoting cell proliferation and resistance to apoptosis, as well as tumor angiogenesis, invasion, and migration. In the characterization of effects of ST3-H2A2, a selective inhibitor of the STAT3 N-terminal domain (ND), we observed that the compound induced apoptotic death in cancer cells associated with robust activation of proapoptotic genes. Using ChIP and tiling human promoter arrays, we found that activation of gene expression in response to ST3-H2A2 is accompanied by altered STAT3 chromatin binding. Using inhibitors of STAT3 phosphorylation and a dominant-negative STAT3 mutant, we found that the unphosphorylated form of STAT3 binds to regulatory regions of proapoptotic genes and prevents their expression in tumor cells but not normal cells. siRNA knockdown confirmed the effects of ST3-HA2A on gene expression and chromatin binding to be STAT3 dependent. The STAT3-binding region of the C/EBP-homologous protein (CHOP) promoter was found to be localized in DNaseI hypersensitive site of chromatin in cancer cells but not in nontransformed cells, suggesting that STAT3 binding and suppressive action can be chromatin structure dependent. These data demonstrate a suppressive role for the STAT3 ND in the regulation of proapoptotic gene expression in cancer cells, providing further support for targeting STAT3 ND for cancer therapy.

Microglial activation and antioxidant responses induced by the Parkinson's disease protein α-synuclein.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder typified by tremor, rigidity, akinesia and postural instability due in part to the loss of dopamine within the nigrostriatal system. The pathologic features of this disorder include the loss of substantia nigra dopamine neurons and attendant striatal terminals, the presence of large protein-rich neuronal inclusions containing fibrillar α-synuclein and increased numbers of activated microglia. Evidence suggests that both misfolded α-synuclein and oxidative stress play an important role in the pathogenesis of sporadic PD. Here we review evidence that α-synuclein activates glia inducing inflammation and that Nrf2-directed phase-II antioxidant enzymes play an important role in PD. We also provide new evidence that the expression of antioxidant enzymes regulated in part by Nrf2 is increased in a mouse model of α-synuclein overexpression. We show that misfolded α-synuclein directly activates microglia inducing the production and release of the proinflammatory cytokine, TNF-α, and increasing antioxidant enzyme expression. Importantly, we demonstrate that the precise structure of α-synuclein is important for induction of this proinflammatory pathway. This complex α-synuclein-directed glial response highlights the importance of protein misfolding, oxidative stress and inflammation in PD and represents a potential locus for the development of novel therapeutics focused on induction of the Nrf2-directed antioxidant pathway and inhibition of protein misfolding.

Mechanisms of unphosphorylated STAT3 transcription factor binding to DNA.

Phosphorylation of signal transducer and activator of transcription 3 (STAT3) on a single tyrosine residue in response to growth factors, cytokines, interferons, and oncogenes activates its dimerization, translocation to the nucleus, binding to the interferon γ (gamma)-activated sequence (GAS) DNA-binding site and activation of transcription of target genes. STAT3 is constitutively phosphorylated in various cancers and drives gene expression from GAS-containing promoters to promote tumorigenesis. Recently, roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in maintenance of heterochromatin stability. However, the mechanisms underlying U-STAT3 binding to DNA has not been fully investigated. Here, we explore STAT3-DNA interactions by atomic force microscopy (AFM) imaging. We observed that U-STAT3 molecules bind to the GAS DNA-binding site as dimers and monomers. In addition, we observed that U-STAT3 binds to AT-rich DNA sequence sites and recognizes specific DNA structures, such as 4-way junctions and DNA nodes, within negatively supercoiled plasmid DNA. These structures are important for chromatin organization and our data suggest a role for U-STAT3 as a chromatin/genome organizer. Unexpectedly, we found that a C-terminal truncated 67.5-kDa STAT3 isoform recognizes single-stranded spacers within cruciform structures that also have a role in chromatin organization and gene expression. This isoform appears to be abundant in the nuclei of cancer cells and, therefore, may have a role in regulation of gene expression. Taken together, our data highlight novel mechanisms by which U-STAT3 binds to DNA and supports U-STAT3 function as a transcriptional activator and a chromatin/genomic organizer.

α-Synuclein Alters Toll-Like Receptor Expression.

Parkinson's disease, an age-related neurodegenerative disorder, is characterized by the loss of dopamine neurons in the substantia nigra, the accumulation of α-synuclein in Lewy bodies and neurites, and neuroinflammation. While the exact etiology of sporadic Parkinson's disease remains elusive, a growing body of evidence suggests that misfolded α-synuclein promotes inflammation and oxidative stress resulting in neurodegeneration. α-Synuclein has been directly linked to microglial activation in vitro and increased numbers of activated microglia have been reported in an α-synuclein overexpressing mouse model prior to neuronal loss. However, the mechanism by which α-synuclein incites microglial activation has not been fully described. Microglial activation is governed in part, by pattern recognition receptors that detect foreign material and additionally recognize changes in homeostatic cellular conditions. Upon proinflammatory pathway initiation, activated microglia contribute to oxidative stress through release of cytokines, nitric oxide, and other reactive oxygen species, which may adversely impact adjacent neurons. Here we show that microglia are directly activated by α-synuclein in a classical activation pathway that includes alterations in the expression of toll-like receptors. These data suggest that α-synuclein can act as a danger-associated molecular pattern.

Binding kinetics and activity of human poly(ADP-ribose) polymerase-1 on oligo-deoxyribonucleotide substrates.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a mammalian enzyme that attaches long branching chains of ADP-ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP-1 plays an important role in DNA strand-break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single-stranded to double-strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single-strandedness were used for measurements of both PARP-1/DNA binding kinetics and PARP-1's enzyme activities. We found that binding correlated with activity, but was unrelated to single-strandedness of the ODN. Instead, PARP-1 binding and activity were highest on ODNs that modeled a DNA double-strand break (DSB). These results provide support for PARP-1 recognizing and binding DSBs in a manner that is independent of single-stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP-1 binding and PARP-1 auto-poly(ADP-ribosyl)ation activities within the same in vitro system.

Spatial distribution of radiation-induced double-strand breaks in plasmid DNA as resolved by atomic force microscopy.

Atomic force microscopy (AFM) has been used to directly visualize, size and compare the DNA fragments resulting from exposure to low- and high-LET radiation. Double-stranded pUC-19 plasmid ("naked") DNA samples were irradiated by electron-beam or reactor neutron fluxes with doses ranging from 0.9 to 10 kGy. AFM scanning in the tapping mode was used to image and measure the DNA fragment lengths (ranging from a few bp up to 2864 bp long). Double-strand break (DSB) distributions resulting from high-LET neutron and lower-LET electron irradiation revealed a distinct difference between the effects of these two types of radiation: Low-LET radiation-induced DSBs are distributed more uniformly along the DNA, whereas a much larger proportion of neutron-induced DSBs are distributed locally and densely. Furthermore, comparisons with predictions of a random DSB model of radiation damage show that neutron-induced DSBs deviate more from the model than do electron-induced DSBs. In summary, our high-resolution AFM measurements of radiation-induced DNA fragment-length distributions reveal an increased number of very short fragments and hence clustering of DSBs induced by the high-LET neutron radiation compared with low-LET electron radiation and a random DSB model prediction.

DNA transitions induced by binding of PARP-1 to cruciform structures in supercoiled plasmids.

BACKGROUND: Poly(ADP-ribose)polymerase-1 (PARP-1) binds to single and double-stranded breaks in DNA, but less well known is its affinity for undamaged DNA. Previously, we have shown that PARP-1 also binds to the hairpin structures in DNA models. The mechanism underlying these interactions remains to be defined. METHODS: We analyzed atomic force microscopy (AFM) images of complex of PARP-1 proteins with supercoiled plasmids containing cruciform structures. Using volume measurement analysis of molecules of PARP-1, we determined the numbers of PARP-1 molecules interacting with supercoiled DNA plasmids containing one cruciform structure. We also determined the extent of supercoiling of plasmids. RESULTS: Our observations show that PARP-1 binds to sequences that transition from B-DNA to cruciform structures. PARP-1 is present at the ends of hairpin arms, sites containing a 4-base single-stranded DNA. Furthermore, interaction of PARP-1 with supercoiled plasmids leads to a more relaxed plasmid-DNA conformation. CONCLUSIONS: Binding of PARP-1 to cruciform DNA offers insight into possible mechanisms underlying with changes in DNA conformation. These observations may offer insight into mechanisms involving DNA conformation related to process such as DNA repair and transcription.

End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes.

Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine-homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA-DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.

Tailoring the activity of restriction endonuclease PleI by PNA-induced DNA looping.

DNA looping is one of the key factors allowing proteins bound to different DNA sites to signal one another via direct contacts. We demonstrate that DNA looping can be generated in an arbitrary chosen site by sequence-directed targeting of double-stranded DNA with pseudocomplementary peptide-nucleic acids (pcPNAs). We designed pcPNAs to mask the DNA from cleavage by type IIs restriction enzyme PleI while not preventing the enzyme from binding to its primary DNA recognition site. Direct interaction between two protein molecules (one bound to the original recognition site and the other to a sequence-degenerated site) results in a totally new activity of PleI: it produces a nick near the degenerate site. The PNA-induced nicking efficiency varies with the distance between the two protein-binding sites in a phase with the DNA helical periodicity. Our findings imply a general approach for the fine-tuning of proteins bound to DNA sites well separated along the DNA chain.

Transcriptional repression by binding of poly(ADP-ribose) polymerase to promoter sequences.

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme that plays roles in response to DNA damage, apoptosis, and genetic stability. Recent evidence has implicated PARP in transcription of eukaryotic genes. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene-regulating domains in the absence of DNA damage. Here we report that PARP can bind to the DNA secondary structures (hairpins) in heteroduplex DNA in a DNA end-independent fashion and that automodification of PARP in the presence of NAD+ inhibited its hairpin binding activity. Atomic force microscopic images show that in vitro PARP protein has a preference for the promoter region of the PARP gene in superhelical DNA where the dyad symmetry elements likely form hairpins according to DNase probing. Using a chromatin cross-linking and immunoprecipitation assay we show that PARP protein binds to the chromosomal PARP promoter in vivo. Reporter gene assays have revealed that the transcriptional activity of the PARP promoter is 4-5-fold greater in PARP knockout cells than in wild type fibroblasts. Reintroduction of vectors expressing full-length PARP protein or its truncated mutant (DNA-binding domain retained but lacking catalytic activity) into PARP(-/-) cells has conferred transcriptional down-regulation of the PARP gene promoter. These data provide support for PARP protein as a potent regulator of transcription including down-regulation of its own promoter.