The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Serhiy Souchelnytskyi - One of the best experts on this subject based on the ideXlab platform.
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TGFβ1/Smad3 counteracts BRCA1-dependent repair of DNA damage
Oncogene, 2005Co-Authors: Anna Dubrovska, Takashi Kanamoto, Marta Lomnytska, Carl-henrik Heldin, Natalya Volodko, Serhiy SouchelnytskyiAbstract:Inactivation of the BRCA1 gene has been found to confer susceptibility to early-onset familial breast and ovarian cancers. BRCA1 regulates DNA repair, chromatin remodeling and affects gene transcription. Transforming growth factor- β (TGF β ) is a potent regulator of growth, apoptosis and invasiveness of tumor cells, including breast cancer cells. Here we show that Smad3 which is a component of the TGF β signaling pathway, forms a complex with BRCA1 in vitro and in vivo . The interaction is mediated by the MH1 Domain of Smad3 and the C-terminal part of BRCA1. We observed a co-localization of Smad3 and BRCA1 in nuclear complexes. We also found that TGF β 1/Smad3 counteracted BRCA1-dependent repair of DNA double-strand breaks in human breast epithelial cells, as evaluated by BRCA1 nuclear foci formation, single-cell gel electrophoresis and cell survival assays. Thus, TGF β 1/Smad3 suppresses BRCA1-dependent DNA repair in response to a DNA damaging agent.
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TGFbeta1/Smad3 counteracts BRCA1-dependent repair of DNA damage.
Oncogene, 2005Co-Authors: Anna Dubrovska, Takashi Kanamoto, Marta Lomnytska, Carl-henrik Heldin, Natalya Volodko, Serhiy SouchelnytskyiAbstract:Inactivation of the BRCA1 gene has been found to confer susceptibility to early-onset familial breast and ovarian cancers. BRCA1 regulates DNA repair, chromatin remodeling and affects gene transcription. Transforming growth factor-β (TGFβ) is a potent regulator of growth, apoptosis and invasiveness of tumor cells, including breast cancer cells. Here we show that Smad3 which is a component of the TGFβ signaling pathway, forms a complex with BRCA1 in vitro and in vivo. The interaction is mediated by the MH1 Domain of Smad3 and the C-terminal part of BRCA1. We observed a co-localization of Smad3 and BRCA1 in nuclear complexes. We also found that TGFβ1/Smad3 counteracted BRCA1-dependent repair of DNA double-strand breaks in human breast epithelial cells, as evaluated by BRCA1 nuclear foci formation, single-cell gel electrophoresis and cell survival assays. Thus, TGFβ1/Smad3 suppresses BRCA1-dependent DNA repair in response to a DNA damaging agent.
Prasanna R Kolatkar - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for the cooperative DNA recognition by Smad4 MH1 dimers
Nucleic Acids Research, 2011Co-Authors: Nithya Baburajendran, Ralf Jauch, Kamesh Narasimhan, Prasanna R KolatkarAbstract:Smad proteins form multimeric complexes consisting of the ‘common partner’ Smad4 and receptor regulated R-Smads on clustered DNA binding sites. Deciphering how pathway specific Smad complexes multimerize on DNA to regulate gene expression is critical for a better understanding of the cis-regulatory logic of TGF-b and BMP signaling. To this end, we solved the crystal structure of the dimeric Smad4 MH1 Domain bound to a palindromic Smad binding element. Surprisingly, the Smad4 MH1 forms a constitutive dimer on the SBE DNA without exhibiting any direct protein–protein interactions suggesting a DNA mediated indirect readout mechanism. However, the R-Smads Smad1, Smad2 and Smad3 homodimerize with substantially decreased efficiency despite pronounced structural similarities to Smad4. Therefore, intricate variations in the DNA structure induced by different Smads and/or variant energetic profiles likely contribute to their propensity to dimerize on DNA. Indeed, competitive binding assays revealed that the Smad4/R-Smad heterodimers predominate under equilibrium conditions while R-Smad homodimers are least favored. Together, we present the structural basis for DNA recognition by Smad4 and demonstrate that Smad4 constitutively homo- and heterodimerizes on DNA in contrast to its R-Smad partner proteins by a mechanism independent of direct protein contacts.
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Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-β effectors
Nucleic Acids Research, 2010Co-Authors: Nithya Baburajendran, Paaventhan Palasingam, Ralf Jauch, Kamesh Narasimhan, Shyam Prabhakar, Prasanna R KolatkarAbstract:Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-β-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 Domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a Domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.
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crystal optimization and preliminary diffraction data analysis of the smad1 MH1 Domain bound to a palindromic sbe dna element
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009Co-Authors: Nithya Baburajendran, Paaventhan Palasingam, Calista Keow Leng Ng, Ralf Jauch, Prasanna R KolatkarAbstract:The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) Domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 A resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 A, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit.
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Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 Domain bound to a palindromic SBE DNA element.
Acta crystallographica. Section F Structural biology and crystallization communications, 2009Co-Authors: Nithya Baburajendran, Paaventhan Palasingam, Calista Keow Leng Ng, Ralf Jauch, Prasanna R KolatkarAbstract:The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) Domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 angstrom resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 angstrom, alpha = beta = gamma = 90 degrees. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit.
Anna Dubrovska - One of the best experts on this subject based on the ideXlab platform.
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TGFβ1/Smad3 counteracts BRCA1-dependent repair of DNA damage
Oncogene, 2005Co-Authors: Anna Dubrovska, Takashi Kanamoto, Marta Lomnytska, Carl-henrik Heldin, Natalya Volodko, Serhiy SouchelnytskyiAbstract:Inactivation of the BRCA1 gene has been found to confer susceptibility to early-onset familial breast and ovarian cancers. BRCA1 regulates DNA repair, chromatin remodeling and affects gene transcription. Transforming growth factor- β (TGF β ) is a potent regulator of growth, apoptosis and invasiveness of tumor cells, including breast cancer cells. Here we show that Smad3 which is a component of the TGF β signaling pathway, forms a complex with BRCA1 in vitro and in vivo . The interaction is mediated by the MH1 Domain of Smad3 and the C-terminal part of BRCA1. We observed a co-localization of Smad3 and BRCA1 in nuclear complexes. We also found that TGF β 1/Smad3 counteracted BRCA1-dependent repair of DNA double-strand breaks in human breast epithelial cells, as evaluated by BRCA1 nuclear foci formation, single-cell gel electrophoresis and cell survival assays. Thus, TGF β 1/Smad3 suppresses BRCA1-dependent DNA repair in response to a DNA damaging agent.
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TGFbeta1/Smad3 counteracts BRCA1-dependent repair of DNA damage.
Oncogene, 2005Co-Authors: Anna Dubrovska, Takashi Kanamoto, Marta Lomnytska, Carl-henrik Heldin, Natalya Volodko, Serhiy SouchelnytskyiAbstract:Inactivation of the BRCA1 gene has been found to confer susceptibility to early-onset familial breast and ovarian cancers. BRCA1 regulates DNA repair, chromatin remodeling and affects gene transcription. Transforming growth factor-β (TGFβ) is a potent regulator of growth, apoptosis and invasiveness of tumor cells, including breast cancer cells. Here we show that Smad3 which is a component of the TGFβ signaling pathway, forms a complex with BRCA1 in vitro and in vivo. The interaction is mediated by the MH1 Domain of Smad3 and the C-terminal part of BRCA1. We observed a co-localization of Smad3 and BRCA1 in nuclear complexes. We also found that TGFβ1/Smad3 counteracted BRCA1-dependent repair of DNA double-strand breaks in human breast epithelial cells, as evaluated by BRCA1 nuclear foci formation, single-cell gel electrophoresis and cell survival assays. Thus, TGFβ1/Smad3 suppresses BRCA1-dependent DNA repair in response to a DNA damaging agent.
Jiawei Wu - One of the best experts on this subject based on the ideXlab platform.
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structural basis for the smad5 MH1 Domain to recognize different dna sequences
Nucleic Acids Research, 2015Co-Authors: Nan Chai, Wanxin Li, Jue Wang, Zhixin Wang, Shiming Yang, Jiawei WuAbstract:Smad proteins are important intracellular mediators of TGF-β signalling, which transmit signals directly from cell surface receptors to the nucleus. The MH1 Domain of Smad plays a key role in DNA recognition. Two types of DNA sequence were identified as Smad binding motifs: the Smad binding element (SBE) and the GC-rich sequence. Here we report the first crystal structure of the Smad5 MH1 Domain in complex with the GC-rich sequence. Compared with the Smad5-MH1/SBE complex structure, the Smad5 MH1 Domain contacts the GC-rich site with the same β-hairpin, but the detailed interaction modes are different. Conserved β-hairpin residues make base specific contacts with the minimal GC-rich site, 5′-GGC-3′. The assembly of Smad5-MH1 on the GC-rich DNA also results in distinct DNA conformational changes. Moreover, the crystal structure of Smad5-MH1 in complex with a composite DNA sequence demonstrates that the MH1 Domain is targeted to each binding site (GC-rich or SBE) with modular binding modes, and the length of the DNA spacer affects the MH1 assembly. In conclusion, our work provides the structural basis for the recognition and binding specificity of the Smad MH1 Domain with the DNA targets.
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Features of a Smad3 MH1-DNA complex. Roles of water and zinc in DNA binding.
Journal of Biological Chemistry, 2003Co-Authors: Jijie Chai, Jiawei Wu, Joan Massague, Nikola P PavletichAbstract:Abstract The Smad family of proteins mediates transforming growth factor-β signaling from cell membrane to the nucleus. In the nucleus, Smads serve as transcription factors by directly binding to specific DNA sequences and regulating the expression of ligand-response genes. A previous structural analysis, at 2.8-A resolution, revealed a novel DNA-binding mode for the Smad MH1 Domain but did not allow accurate assignment of the fines features of protein-DNA interactions. The crystal structure of a Smad3 MH1 Domain bound to a palindromic DNA sequence, determined at 2.4-A resolution, reveals a surprisingly important role for water molecules. The asymmetric placement of the DNA-binding motif (a conserved 11-residue β-hairpin) in the major groove of DNA is buttressed by seven well ordered water molecules. These water molecules make specific hydrogen bonds to the DNA bases, the DNA phosphate backbones, and several critical Smad3 residues. In addition, the MH1 Domain is found to contain a bound zinc atom using four invariant residues among Smad proteins, three cysteines and one histidine. Removal of the zinc atom results in compromised DNA binding activity. These results define the Smad MH1 Domain as a zinc-coordinating module that exhibits unique DNA binding properties.
Nithya Baburajendran - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for the cooperative DNA recognition by Smad4 MH1 dimers
Nucleic Acids Research, 2011Co-Authors: Nithya Baburajendran, Ralf Jauch, Kamesh Narasimhan, Prasanna R KolatkarAbstract:Smad proteins form multimeric complexes consisting of the ‘common partner’ Smad4 and receptor regulated R-Smads on clustered DNA binding sites. Deciphering how pathway specific Smad complexes multimerize on DNA to regulate gene expression is critical for a better understanding of the cis-regulatory logic of TGF-b and BMP signaling. To this end, we solved the crystal structure of the dimeric Smad4 MH1 Domain bound to a palindromic Smad binding element. Surprisingly, the Smad4 MH1 forms a constitutive dimer on the SBE DNA without exhibiting any direct protein–protein interactions suggesting a DNA mediated indirect readout mechanism. However, the R-Smads Smad1, Smad2 and Smad3 homodimerize with substantially decreased efficiency despite pronounced structural similarities to Smad4. Therefore, intricate variations in the DNA structure induced by different Smads and/or variant energetic profiles likely contribute to their propensity to dimerize on DNA. Indeed, competitive binding assays revealed that the Smad4/R-Smad heterodimers predominate under equilibrium conditions while R-Smad homodimers are least favored. Together, we present the structural basis for DNA recognition by Smad4 and demonstrate that Smad4 constitutively homo- and heterodimerizes on DNA in contrast to its R-Smad partner proteins by a mechanism independent of direct protein contacts.
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Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-β effectors
Nucleic Acids Research, 2010Co-Authors: Nithya Baburajendran, Paaventhan Palasingam, Ralf Jauch, Kamesh Narasimhan, Shyam Prabhakar, Prasanna R KolatkarAbstract:Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-β-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 Domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a Domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.
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crystal optimization and preliminary diffraction data analysis of the smad1 MH1 Domain bound to a palindromic sbe dna element
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009Co-Authors: Nithya Baburajendran, Paaventhan Palasingam, Calista Keow Leng Ng, Ralf Jauch, Prasanna R KolatkarAbstract:The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) Domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 A resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 A, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit.
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Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 Domain bound to a palindromic SBE DNA element.
Acta crystallographica. Section F Structural biology and crystallization communications, 2009Co-Authors: Nithya Baburajendran, Paaventhan Palasingam, Calista Keow Leng Ng, Ralf Jauch, Prasanna R KolatkarAbstract:The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) Domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 angstrom resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 angstrom, alpha = beta = gamma = 90 degrees. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit.