24 research outputs found
Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide
Pyrrole-imidazole (Py-Im) polyamides are high affinity DNA-binding small molecules that can inhibit protein-DNA interactions. In VCaP cells, a human prostate cancer cell line overexpressing both AR and the TMPRSS2-ERG gene fusion, an androgen response element (ARE)-targeted Py-Im polyamide significantly downregulates AR driven gene expression. Polyamide exposure to VCaP cells reduced proliferation without causing DNA damage. Py-Im polyamide treatment also reduced tumor growth in a VCaP mouse xenograft model. In addition to the effects on AR regulated transcription, RNA-seq analysis revealed inhibition of topoisomerase-DNA binding as a potential mechanism that contributes to the antitumor effects of polyamides in cell culture and in xenografts. These studies support the therapeutic potential of Py-Im polyamides to target multiple aspects of transcriptional regulation in prostate cancers without genotoxic stress
RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif
RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a āminor groove sensor.ā Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy
Structural basis for the initiation of eukaryotic transcription-coupled DNA repair
Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity1. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol IIāCSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol IIāRad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation
RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif
RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a āminor groove sensor.ā Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy
Structural basis for the initiation of eukaryotic transcription-coupled DNA repair
Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity1. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol IIāCSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol IIāRad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation
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Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide.
Pyrrole-imidazole (Py-Im) polyamides are high affinity DNA-binding small molecules that can inhibit protein-DNA interactions. In VCaP cells, a human prostate cancer cell line overexpressing both AR and the TMPRSS2-ERG gene fusion, an androgen response element (ARE)-targeted Py-Im polyamide significantly downregulates AR driven gene expression. Polyamide exposure to VCaP cells reduced proliferation without causing DNA damage. Py-Im polyamide treatment also reduced tumor growth in a VCaP mouse xenograft model. In addition to the effects on AR regulated transcription, RNA-seq analysis revealed inhibition of topoisomerase-DNA binding as a potential mechanism that contributes to the antitumor effects of polyamides in cell culture and in xenografts. These studies support the therapeutic potential of Py-Im polyamides to target multiple aspects of transcriptional regulation in prostate cancers without genotoxic stress
Recommended from our members
Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide
Pyrrole-imidazole (Py-Im) polyamides are high affinity DNA-binding small molecules that can inhibit protein-DNA interactions. In VCaP cells, a human prostate cancer cell line overexpressing both AR and the TMPRSS2-ERG gene fusion, an androgen response element (ARE)-targeted Py-Im polyamide significantly downregulates AR driven gene expression. Polyamide exposure to VCaP cells reduced proliferation without causing DNA damage. Py-Im polyamide treatment also reduced tumor growth in a VCaP mouse xenograft model. In addition to the effects on AR regulated transcription, RNA-seq analysis revealed inhibition of topoisomerase-DNA binding as a potential mechanism that contributes to the antitumor effects of polyamides in cell culture and in xenografts. These studies support the therapeutic potential of Py-Im polyamides to target multiple aspects of transcriptional regulation in prostate cancers without genotoxic stress
LASER SPECTROSCOPY OF A DOUBLY-SUBSTITUTED AROMATIC WITH TWO FLEXIBLE SIDE CHAINS: INTRACHAIN AND INTERCHAIN CONFORMATIONAL PREFERENCES
Author Institution: Department of Chemistry, Purdue University, West Lafayette, IN 47907; Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260Building on previous studies of the spectroscopy and isomerization dynamics of small biologically relevant molecules, we have been motivated to study molecules designed to be prototypical of different types of potential energy landscapes. In that spirit, O-acetamidoethyl-N-acetyltyramine (OANAT) possesses two flexible side chains of similar size, substituted at opposite ends of a phenyl ring, which intuitively should have two largely decoupled regions of the potential energy surface involving coordinates localized on one or the other side chain. In addition, the chains have sufficient length and H-bonding capacity to fold back over the phenyl ring, forming interchain hydrogen bonds. Resonant two-photon ionization and UV-UV holeburning spectroscopy identifies six conformational isomers of OANAT. Resonant ion-dip infrared spectroscopy identified three of these six as independent chain conformers, and three as possessing interchain H-bonds. To better understand the independent chain class, single chain molecules containing one or the other peptide mimetic side chains of OANAT were also studied. These molecules include the alkyl chain species N-phenethyl-acetamide (NPEA, 1 conformer) and N-(p-methoxyphenethyl-acetamide) (NMPEA, 3 conformers), and the alkoxy chain species N-(2-phenoxyethyl)-acetamide (NPOEA, 4 conformers). The electronic and infrared spectra for these molecules are presented along with DFT, TDDFT, MP2, RIMP2, and RICC2 calculations. The S-S conformer origins of OANAT and NPOEA were spread over 1000 and 750 \wn, respectively, encouraging use of relative origin position as a diagnostic for conformational assignment. Based on the full body of spectroscopic evidence, structural assignments for these molecules are made and will be discussed
Virtual Screening and Optimization Yield Low-Nanomolar Inhibitors of the Tautomerase Activity of <i>Plasmodium falciparum</i> Macrophage Migration Inhibitory Factor
The <i>Plasmodium falciparum</i> orthologue
of the human cytokine, macrophage migratory inhibitory factor (<i>Pf</i>MIF), is produced by the parasite during malaria infection
and modulates the hostās immune response. As for other MIF
orthologues, <i>Pf</i>MIF has tautomerase activity, whose
inhibition may influence the cytokine activity. To identify small-molecule
inhibitors of the tautomerase activity of <i>Pf</i>MIF,
virtual screening has been performed by docking 2.1 million compounds
into the enzymatic site. Assaying of 17 compounds identified four
as active. Substructure search for the most potent of these compounds,
a 4-phenoxypyridine analogue, identified four additional compounds
that were purchased and also shown to be active. Thirty-one additional
analogues were then designed, synthesized, and assayed. Three were
found to be potent <i>Pf</i>MIF tautomerase inhibitors with <i>K</i><sub>i</sub> of ā¼40 nM; they are also highly selective
with <i>K</i><sub>i</sub> > 100 Ī¼M for human MIF