The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Bruce Stillman - One of the best experts on this subject based on the ideXlab platform.
-
cdc6 induced conformational changes in orc bound to origin dna revealed by cryo electron microscopy
Structure, 2012Co-Authors: Jingchuan Sun, Hironori Kawakami, Juergen Zech, Christian Speck, Bruce StillmanAbstract:The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH Domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.
-
structural basis for origin recognition complex 1 protein silence information regulator 1 protein interaction in epigenetic silencing
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bruce Stillman, Ruiming XuAbstract:The interaction between silence information regulator 1 protein (Sir1p) and origin recognition complex 1 protein (Orc1p), the largest subunit of the origin recognition complex, plays an important role in the establishment of transcriptional silencing at the cryptic mating-type gene loci in Saccharomyces cerevisiae. Sir1p binds the N-terminal region of Orc1p encompassing a Bromo-adjacent homology (BAH) Domain found in various chromatin-associated proteins. To understand the molecular mechanism of Sir protein recruitment, we have determined a 2.5-A cocrystal structure of the N-terminal Domain of Orc1p in complex with the Orc1p-interacting Domain of Sir1p. The structure reveals that Sir1p Orc1p-interacting Domain has a bilobal structure: an α/β N-terminal lobe and a C-terminal lobe resembling the Tudor Domain royal family fold. The N-terminal lobe of Sir1p binds in a shallow groove between a helical subDomain and the BAH Domain of Orc1p. The structure provides a mechanistic understanding of Orc1p–Sir1p interaction specificity, as well as insights into protein–protein interactions involving BAH Domains in general.
-
Structural basis for origin recognition complex 1 protein–silence information regulator 1 protein interaction in epigenetic silencing
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Hao-chi Hsu, Bruce StillmanAbstract:The interaction between silence information regulator 1 protein (Sir1p) and origin recognition complex 1 protein (Orc1p), the largest subunit of the origin recognition complex, plays an important role in the establishment of transcriptional silencing at the cryptic mating-type gene loci in Saccharomyces cerevisiae. Sir1p binds the N-terminal region of Orc1p encompassing a Bromo-adjacent homology (BAH) Domain found in various chromatin-associated proteins. To understand the molecular mechanism of Sir protein recruitment, we have determined a 2.5-A cocrystal structure of the N-terminal Domain of Orc1p in complex with the Orc1p-interacting Domain of Sir1p. The structure reveals that Sir1p Orc1p-interacting Domain has a bilobal structure: an α/β N-terminal lobe and a C-terminal lobe resembling the Tudor Domain royal family fold. The N-terminal lobe of Sir1p binds in a shallow groove between a helical subDomain and the BAH Domain of Orc1p. The structure provides a mechanistic understanding of Orc1p–Sir1p interaction specificity, as well as insights into protein–protein interactions involving BAH Domains in general.
-
Structure and function of the BAH-containing Domain of Orc1p in epigenetic silencing.
The EMBO journal, 2002Co-Authors: Zhiguo Zhang, Mariko K. Hayashi, Olaf Merkel, Bruce StillmanAbstract:The N‐terminal Domain of the largest subunit of the Saccharomyces cerevisiae origin recognition complex (Orc1p) functions in transcriptional silencing and contains a bromo‐adjacent homology (BAH) Domain found in some chromatin‐associated proteins including Sir3p. The 2.2 A crystal structure of the N‐terminal Domain of Orc1p revealed a BAH core and a non‐conserved helical sub‐Domain. Mutational analyses demonstrated that the helical sub‐Domain was necessary and sufficient to bind Sir1p, and critical for targeting Sir1p primarily to the cis ‐acting E silencers at the HMR and HML silent chromatin Domains. In the absence of the BAH Domain, ∼14–20% of cells in a population were silenced at the HML locus. Moreover, the distributions of the Sir2p, Sir3p and Sir4p proteins, while normal, were at levels lower than found in wild‐type cells. Thus, in the absence of the Orc1p BAH Domain, HML resembled silencing of genes adjacent to telomeres. These data are consistent with the view that the Orc1p–Sir1p interaction at the E silencers ensures stable inheritance of pre‐established Sir2p, Sir3p and Sir4p complexes at the silent mating type loci.
Danesh Moazed - One of the best experts on this subject based on the ideXlab platform.
-
Heterochromatin protein Sir3 induces contacts between the amino terminus of histone H4 and nucleosomal DNA
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Feng Wang, Mohammed Altaf, Mark A. Currie, Aaron M. Johnson, Danesh MoazedAbstract:The regulated binding of effector proteins to the nucleosome plays a central role in the activation and silencing of eukaryotic genes. How this binding changes the properties of chromatin to mediate gene activation or silencing is not fully understood. Here we provide evidence that association of the budding yeast silent information regulator 3 (Sir3) silencing protein with the nucleosome induces a conformational change in the amino terminus of histone H4 that promotes interactions between the conserved H4 arginines 17 and 19 (R17 and R19) and nucleosomal DNA. Substitutions of H4R17 and R19 with alanine abolish silencing in vivo, but have little or no effect on binding of Sir3 to nucleosomes or histone H4 peptides in vitro. Furthermore, in both the previously reported crystal structure of the Sir3-bromo adjacent homology (BAH) Domain bound to the Xenopus laevis nucleosome core particle and the crystal structure of the Sir3-BAH Domain bound to the yeast nucleosome core particle described here, H4R17 and R19 make contacts with nucleosomal DNA rather than with Sir3. These results suggest that Sir3 binding generates a more stable nucleosome by clamping H4R17 and R19 to nucleosomal DNA, and raise the possibility that such induced changes in histone–DNA contacts play major roles in the regulation of chromatin structure.
-
Sir3-Nucleosome Interactions in Spreading of Silent Chromatin in Saccharomyces cerevisiae
Molecular and cellular biology, 2008Co-Authors: Johannes R. Buchberger, Megumi Onishi, Jan Seebacher, Adam D. Rudner, Steven P. Gygi, Danesh MoazedAbstract:Silent chromatin in Saccharomyces cerevisiae is established in a stepwise process involving the SIR complex, comprised of the histone deacetylase Sir2 and the structural components Sir3 and Sir4. The Sir3 protein, which is the primary histone-binding component of the SIR complex, forms oligomers in vitro and has been proposed to mediate the spreading of the SIR complex along the chromatin fiber. In order to analyze the role of Sir3 in the spreading of the SIR complex, we performed a targeted genetic screen for alleles of SIR3 that dominantly disrupt silencing. Most mutations mapped to a single surface in the conserved N-terminal BAH Domain, while one, L738P, localized to the AAA ATPase-like Domain within the C-terminal half of Sir3. The BAH point mutants, but not the L738P mutant, disrupted the interaction between Sir3 and nucleosomes. In contrast, Sir3-L738P bound the N-terminal tail of histone H4 more strongly than wild-type Sir3, indicating that misregulation of the Sir3 C-terminal histone-binding activity also disrupted spreading. Our results underscore the importance of proper interactions between Sir3 and the nucleosome in silent chromatin assembly. We propose a model for the spreading of the SIR complex along the chromatin fiber through the two distinct histone-binding Domains in Sir3.
-
Role of the Conserved Sir3-BAH Domain in Nucleosome Binding and Silent Chromatin Assembly
Molecular cell, 2007Co-Authors: Megumi Onishi, Gunn-guang Liou, Johannes R. Buchberger, Thomas Walz, Danesh MoazedAbstract:Silent chromatin Domains in Saccharomyces cerevisiae represent examples of epigenetically heritable chromatin. The formation of these Domains involves the recruitment of the SIR complex, composed of Sir2, Sir3, and Sir4, followed by iterative cycles of NAD-dependent histone deacetylation and spreading of SIR complexes over adjacent chromatin Domains. We show here that the conserved bromo-adjacent homology (BAH) Domain of Sir3 is a nucleosome- and histone-tail-binding Domain and that its binding to nucleosomes is regulated by residues in the N terminus of histone H4 and the globular Domain of histone H3 on the exposed surface of the nucleosome. Furthermore, using a partially purified system containing nucleosomes, the three Sir proteins, and NAD, we observe the formation of SIR-nucleosome filaments with a diameter of less than 20 nm. Together, these observations suggest that the SIR complex associates with an extended chromatin fiber through interactions with two different regions in the nucleosome.
Jessica A. Downs - One of the best experts on this subject based on the ideXlab platform.
-
The BAH Domain of BAF180 is required for PCNA ubiquitination.
Mutation research, 2015Co-Authors: Atsuko Niimi, Jessica A. Downs, Suzanna R. Hopkins, Chikahide MasutaniAbstract:Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of post replication repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) Domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH Domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH Domains are sufficient to promote PCNA ubiquitination.
-
The BAH Domain of Rsc2 is a histone H3 binding Domain
Nucleic acids research, 2013Co-Authors: Anna L Chambers, Antony W. Oliver, Laurence H. Pearl, Jessica A. DownsAbstract:Bromo-adjacent homology (BAH) Domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH Domains bind nucleosomes, the binding partners of RSC-like BAH Domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH Domain and, like the Sir3-like BAH Domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH Domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH Domain, we determined its crystal structure at 2.4 A resolution. We find that it differs substantially from Sir3-like BAH Domains and lacks the motifs in these Domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH Domains and identify a novel motif that mediates this interaction.
-
The Two Different Isoforms of the RSC Chromatin Remodeling Complex Play Distinct Roles in DNA Damage Responses
2013Co-Authors: Anna L Chambers, Peter M. Brownlee, Samuel C. Durley, Tracey Beacham, Nicholas A. Kent, Jessica A. DownsAbstract:The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the Domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH Domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarit
-
Rsc1 Domains can functionally compensate for Rsc2 Domains in DNA damage survival assays.
2013Co-Authors: Anna L Chambers, Peter M. Brownlee, Samuel C. Durley, Tracey Beacham, Nicholas A. Kent, Jessica A. DownsAbstract:(A) Cartoon of Domain swapped constructs used in the assays. BromoDomain 1 (BD1), bromoDomain 2 (BD2) or the bromoadjacent homology (BAH) Domain of Rsc2 was replaced with the analogous Domain from Rsc1. (B) Serial dilutions of mid-log cultures of wt with vector alone or rsc2 mutant strain containing vector alone (pRS415), pRSC2, or Domain swap expression plasmids were plated onto media containing the indicated dose of MMS. (C) Serial dilutions of mid-log cultures of wt with vector alone, rsc1 with vector alone, or rsc1/rsc2 mutant strain with pRSC2 or Domain swap expression plasmids were plated onto media containing the indicated dose of MMS. (D) Survival of strains as in (B) in the presence of continued transcriptional induction of the HO endonuclease. Survival is calculated as the number of colonies surviving in continued HO expression relative to conditions where HO is not expressed. (E) Survival of strains as in (C) in the presence of continued transcriptional induction of the HO endonuclease. Survival is calculated as the number of colonies surviving in continued HO expression relative to conditions where HO is not expressed.
-
The two different isoforms of the RSC chromatin remodeling complex play distinct roles in DNA damage responses
PloS one, 2012Co-Authors: Anna L Chambers, Peter M. Brownlee, Samuel C. Durley, Tracey Beacham, Nicholas A. Kent, Jessica A. DownsAbstract:The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the Domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH Domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarity between Rsc1 and Rsc2, the two different isoforms of RSC provide distinct functions in DNA damage responses, and that at least part of the functional specificity is dictated by the BAH Domains.
Hao-chi Hsu - One of the best experts on this subject based on the ideXlab platform.
-
Structure and function of the Saccharomyces cerevisiae Sir3 BAH Domain
Molecular and cellular biology, 2006Co-Authors: Jessica J. Connelly, Peihua Yuan, Hao-chi Hsu, Rolf SternglanzAbstract:Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homology) Domain at its N terminus. Overexpression of this Domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH Domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH Domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH Domains. The structure of the Sir3 BAH Domain has been determined. In the crystal, the molecule multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH Domain of Sir3 in silencing.
-
Structural basis for origin recognition complex 1 protein–silence information regulator 1 protein interaction in epigenetic silencing
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Hao-chi Hsu, Bruce StillmanAbstract:The interaction between silence information regulator 1 protein (Sir1p) and origin recognition complex 1 protein (Orc1p), the largest subunit of the origin recognition complex, plays an important role in the establishment of transcriptional silencing at the cryptic mating-type gene loci in Saccharomyces cerevisiae. Sir1p binds the N-terminal region of Orc1p encompassing a Bromo-adjacent homology (BAH) Domain found in various chromatin-associated proteins. To understand the molecular mechanism of Sir protein recruitment, we have determined a 2.5-A cocrystal structure of the N-terminal Domain of Orc1p in complex with the Orc1p-interacting Domain of Sir1p. The structure reveals that Sir1p Orc1p-interacting Domain has a bilobal structure: an α/β N-terminal lobe and a C-terminal lobe resembling the Tudor Domain royal family fold. The N-terminal lobe of Sir1p binds in a shallow groove between a helical subDomain and the BAH Domain of Orc1p. The structure provides a mechanistic understanding of Orc1p–Sir1p interaction specificity, as well as insights into protein–protein interactions involving BAH Domains in general.
Fabrizio Martino - One of the best experts on this subject based on the ideXlab platform.
-
The N-terminal acetylation of Sir3 stabilizes its binding to the nucleosome core particle
Nature Structural & Molecular Biology, 2013Co-Authors: Nadia Arnaudo, Israel S Fernández, Stephen H Mclaughlin, Sew Y Peak-chew, Daniela Rhodes, Fabrizio MartinoAbstract:N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH Domain of Sir3 bound to the nucleosome core particle revealed that N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH Domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Additionally, we present a new method for the production of protein–nucleosome complexes for structural analysis.
-
The N-terminal acetylation of Sir3 stabilizes its binding to the nucleosome core particle
Nature structural & molecular biology, 2013Co-Authors: Nadia Arnaudo, Israel S Fernández, Stephen H Mclaughlin, Sew Y Peak-chew, Daniela Rhodes, Fabrizio MartinoAbstract:The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH Domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Additionally, we present a new method for the production of protein-nucleosome complexes for structural analysis.
