43 results on '"Alain Verreault"'
Search Results
2. Unorthodox PCNA Binding by Chromatin Assembly Factor 1
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Amogh Gopinathan Nair, Nick Rabas, Sara Lejon, Caleb Homiski, Michael J. Osborne, Normand Cyr, Aleksandr Sverzhinsky, Thomas Melendy, John M. Pascal, Ernest D. Laue, Katherine L. B. Borden, James G. Omichinski, and Alain Verreault
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DNA Replication ,Organic Chemistry ,General Medicine ,DNA ,Saccharomyces cerevisiae ,Arginine ,Catalysis ,Chromatin ,Computer Science Applications ,Inorganic Chemistry ,Chromatin Assembly Factor-1 ,Proliferating Cell Nuclear Antigen ,CHAF1A ,PCNA ,PIP-Box ,DNA replication ,CAF-1 p150 ,chromatin assembly ,SEC-SAXS ,NMR ,Humans ,Physical and Theoretical Chemistry ,Amino Acids ,Peptides ,Molecular Biology ,Spectroscopy - Abstract
The eukaryotic DNA replication fork is a hub of enzymes that continuously act to synthesize DNA, propagate DNA methylation and other epigenetic marks, perform quality control, repair nascent DNA, and package this DNA into chromatin. Many of the enzymes involved in these spatiotemporally correlated processes perform their functions by binding to proliferating cell nuclear antigen (PCNA). A long-standing question has been how the plethora of PCNA-binding enzymes exert their activities without interfering with each other. As a first step towards deciphering this complex regulation, we studied how Chromatin Assembly Factor 1 (CAF-1) binds to PCNA. We demonstrate that CAF-1 binds to PCNA in a heretofore uncharacterized manner that depends upon a cation-pi (π) interaction. An arginine residue, conserved among CAF-1 homologs but absent from other PCNA-binding proteins, inserts into the hydrophobic pocket normally occupied by proteins that contain canonical PCNA interaction peptides (PIPs). Mutation of this arginine disrupts the ability of CAF-1 to bind PCNA and to assemble chromatin. The PIP of the CAF-1 p150 subunit resides at the extreme C-terminus of an apparent long α-helix (119 amino acids) that has been reported to bind DNA. The length of that helix and the presence of a PIP at the C-terminus are evolutionarily conserved among numerous species, ranging from yeast to humans. This arrangement of a very long DNA-binding coiled-coil that terminates in PIPs may serve to coordinate DNA and PCNA binding by CAF-1.
- Published
- 2022
3. Mechanisms to reduce the cytotoxicity of pharmacological nicotinamide concentrations in the pathogenic fungus Candida albicans
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Rahul Ghugari, Alain Verreault, Mark S. Schmidt, Charles Brenner, Eric Bonneil, and Sarah Tsao
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Niacinamide ,0301 basic medicine ,Saccharomyces cerevisiae Proteins ,animal structures ,Cell Survival ,Biochemistry ,Histone Deacetylases ,Microbiology ,Fungal Proteins ,Histones ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Candida albicans ,Humans ,Sirtuins ,Cytotoxicity ,Molecular Biology ,health care economics and organizations ,Cell Proliferation ,biology ,Nicotinamide ,Lysine ,Candidiasis ,food and beverages ,Cell Biology ,NAD ,biology.organism_classification ,Corpus albicans ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Sirtuin ,biology.protein ,Protein deacetylase ,NAD+ kinase ,Intracellular - Abstract
Candida albicans is a pathogenic fungus that causes systemic infections and mortality in immunosuppressed individuals. We previously showed that deacetylation of histone H3 lysine 56 by Hst3 is essential for C. albicans viability. Hst3 is a fungal-specific NAD+ -dependent protein deacetylase of the sirtuin family. In vivo, supraphysiological concentrations of nicotinamide (NAM) are required for Hst3 inhibition and cytotoxicity. This underscores the importance of identifying mechanisms by which C. albicans can modulate intracellular NAM concentrations. For the first time in a pathogenic fungus, we combine genetics, heavy isotope labeling, and targeted quantitative metabolomics to identify genes, pathways, and mechanisms by which C. albicans can reduce the cytotoxicity of high NAM concentrations. We discovered three distinct fates for supraphysiological NAM concentrations. First, upon transient exposure to NAM, high intracellular NAM concentrations rapidly return near the physiological levels observed in cells that are not exposed to NAM. Second, during the first step of a fungal-specific NAM salvage pathway, NAM is converted into nicotinic acid, a metabolite that cannot inhibit the sirtuin Hst3. Third, we provide evidence that NAM enters the NAD+ metabolome through a NAM exchange reaction that contributes to NAM-mediated inhibition of sirtuins. However, in contrast to the other fates of NAM, the NAM exchange reaction cannot cause a net decrease in the intracellular concentration of NAM. Therefore, this reaction cannot enhance resistance to NAM. In summary, we demonstrate that C. albicans possesses at least two mechanisms to attenuate the cytotoxicity of pharmacological NAM concentrations. It seems likely that those two mechanisms of resistance to cytotoxic NAM concentrations are conserved in many other pathogenic fungi.
- Published
- 2020
4. Chromatin dynamics and DNA replication roadblocks
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Alain Verreault, Hugo Wurtele, and Ian Hammond-Martel
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DNA Replication ,DNA Repair ,DNA damage ,DNA repair ,Biochemistry ,Chromatin Assembly ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Humans ,Nascent dna ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,DNA replication ,Eukaryota ,DNA ,Cell Biology ,Chromatin Assembly and Disassembly ,Chromatin ,Cell biology ,Histone Code ,Histone ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,DNA Damage - Abstract
A broad spectrum of spontaneous and genotoxin-induced DNA lesions impede replication fork progression. The DNA damage response that acts to promote completion of DNA replication is associated with dynamic changes in chromatin structure that include two distinct processes which operate genome-wide during S-phase. The first, often referred to as histone recycling or parental histone segregation, is characterized by the transfer of parental histones located ahead of replication forks onto nascent DNA. The second, known as de novo chromatin assembly, consists of the deposition of new histone molecules onto nascent DNA. Because these two processes occur at all replication forks, their potential to influence a multitude of DNA repair and DNA damage tolerance mechanisms is considerable. The purpose of this review is to provide a description of parental histone segregation and de novo chromatin assembly, and to illustrate how these processes influence cellular responses to DNA replication roadblocks.
- Published
- 2021
5. Acetylation of PCNA Sliding Surface by Eco1 Promotes Genome Stability through Homologous Recombination
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Jean-François Couture, Alain Verreault, Joseph S. Brunzelle, Anne-Claude Gingras, Jean-Philippe Lambert, Jacques Côté, Pierre Billon, Véronique Tremblay, Jian Li, Tomohiko Sugiyama, and Yizhang Chen
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Models, Molecular ,0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Genotype ,Protein Conformation ,DNA repair ,DNA damage ,Saccharomyces cerevisiae ,Chromatids ,Genomic Instability ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Acetyltransferases ,Proliferating Cell Nuclear Antigen ,Humans ,Molecular Biology ,DNA Polymerase III ,DNA clamp ,030102 biochemistry & molecular biology ,biology ,Lysine ,DNA replication ,Nuclear Proteins ,Recombinational DNA Repair ,Acetylation ,Cell Biology ,Molecular biology ,Proliferating cell nuclear antigen ,Cell biology ,Phenotype ,030104 developmental biology ,chemistry ,Mutation ,biology.protein ,DNA mismatch repair ,Chromosomes, Fungal ,Homologous recombination ,Protein Processing, Post-Translational ,DNA ,DNA Damage - Abstract
During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.
- Published
- 2017
6. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin
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Pierre Thibault, Zhiying You, Alain Verreault, Marlene Gharib, Krassimir Yankulov, Hisao Masai, Naoko Kakusho, Erin Drury, Daniel Jeffery, Brandon A. Wyse, and Michael Weinreich
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DNA Replication ,Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,Biology ,Chromatin remodeling ,S Phase ,Proliferating Cell Nuclear Antigen ,Nucleosome ,Amino Acid Sequence ,Gene Silencing ,Chromatin Assembly Factor-1 ,Phosphorylation ,Molecular Biology ,Cyclin-dependent kinase 1 ,DNA clamp ,Chromatin Assembly Factor I ,Cell Biology ,Telomere ,Chromatin Assembly and Disassembly ,Molecular biology ,Chromatin ,Proliferating cell nuclear antigen ,Mutation ,biology.protein ,CDC28 Protein Kinase, S cerevisiae ,Protein Binding ,Reports ,Developmental Biology - Abstract
Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.
- Published
- 2015
7. Regulation of the Histone Deacetylase Hst3 by Cyclin-dependent Kinases and the Ubiquitin Ligase SCFCdc4
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Pierre Thibault, Xiaojing Tang, Mike Tyers, Evgeny Kanshin, Alain Verreault, Elizabeth C. Williams, Neda Delgoshaie, and Adam D. Rudner
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Saccharomyces cerevisiae Proteins ,Ubiquitin-Protein Ligases ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,DNA and Chromosomes ,Biochemistry ,Histone Deacetylases ,Histones ,Histone H3 ,Cyclin-dependent kinase ,Enzyme Stability ,Phosphorylation ,Molecular Biology ,Cyclin-dependent kinase 1 ,biology ,F-Box Proteins ,Cell Cycle ,Ubiquitination ,Acetylation ,Cell Biology ,Cell cycle ,HDAC4 ,Ubiquitin ligase ,biology.protein ,Histone deacetylase ,Genome, Fungal - Abstract
In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is a modification of new H3 molecules deposited throughout the genome during S-phase. H3K56ac is removed by the sirtuins Hst3 and Hst4 at later stages of the cell cycle. Previous studies indicated that regulated degradation of Hst3 plays an important role in the genome-wide waves of H3K56 acetylation and deacetylation that occur during each cell cycle. However, little is known regarding the mechanism of cell cycle-regulated Hst3 degradation. Here, we demonstrate that Hst3 instability in vivo is dependent upon the ubiquitin ligase SCF(Cdc4) and that Hst3 is phosphorylated at two Cdk1 sites, threonine 380 and threonine 384. This creates a diphosphorylated degron that is necessary for Hst3 polyubiquitylation by SCF(Cdc4). Mutation of the Hst3 diphospho-degron does not completely stabilize Hst3 in vivo, but it nonetheless results in a significant fitness defect that is particularly severe in mutant cells treated with the alkylating agent methyl methanesulfonate. Unexpectedly, we show that Hst3 can be degraded between G2 and anaphase, a window of the cell cycle where Hst3 normally mediates genome-wide deacetylation of H3K56. Our results suggest an intricate coordination between Hst3 synthesis, genome-wide H3K56 deacetylation by Hst3, and cell cycle-regulated degradation of Hst3 by cyclin-dependent kinases and SCF(Cdc4).
- Published
- 2014
8. Unraveling Site-Specific and Combinatorial Histone Modifications Using High-Resolution Mass Spectrometry in Histone Deacetylase Mutants of Fission Yeast
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Alain Verreault, Roshan Elizabeth Rajan, Pierre Thibault, and Nebiyu Abshiru
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0301 basic medicine ,Histone deacetylase 5 ,Histone deacetylase 2 ,Acetylation ,General Chemistry ,Biology ,SAP30 ,Biochemistry ,Molecular biology ,Methylation ,Histone Deacetylases ,Mass Spectrometry ,3. Good health ,Cell biology ,Histone Code ,Histones ,03 medical and health sciences ,030104 developmental biology ,Histone H1 ,Histone methyltransferase ,Histone H2A ,Mutation ,Histone code ,Histone deacetylase ,Schizosaccharomyces pombe Proteins - Abstract
Histone deacetylases (HDACs) catalyze the removal of acetylation marks from lysine residues on histone and nonhistone substrates. Their activity is generally associated with essential cellular processes such as transcriptional repression and heterochromatin formation. Interestingly, abnormal activity of HDACs has been reported in various types of cancers, which makes them a promising therapeutic target for cancer treatment. In the current study, we aim to understand the mechanisms underlying the function of HDACs using an in-depth quantitative analysis of changes in histone acetylation levels in Schizosaccharomyces pombe (S. pombe) lacking major HDAC activities. We employed a targeted quantitative mass spectrometry approach to profile changes of acetylation and methylation at multiple lysine residues on the N-terminal tail of histones H3 and H4. Our analyses identified a number of histone acetylation sites that are significantly affected by S. pombe HDAC mutations. We discovered that mutation of the Class I HDAC known as Clr6 causes a major increase in the abundance of triacetylated H4 molecules at K5, K8, and K12. A clr6-1 hypomorphic mutation also increased the abundance of multiple acetyl-lysines in histone H3. In addition, our study uncovered a few crosstalks between histone acetylation and methylation upon deletion of HDACs Hos2 and Clr3. We anticipate that the results from this study will greatly improve our current understanding of the mechanisms involved in HDAC-mediated gene regulation and heterochromatin assembly.
- Published
- 2016
9. The dual function of LMO2 in driving erythroid cell fate
- Author
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Véronique Lisi, Alain Verreault, François Major, Magali Humbert, Diogo F.T. Veiga, Trang Hoang, Marie-Claude Sincennes, and Bachir Affar
- Subjects
LMO2 ,Cancer Research ,Chemistry ,Genetics ,Erythroid cell ,Cell Biology ,Hematology ,Molecular Biology ,Dual function ,Cell biology - Published
- 2017
10. MYST protein acetyltransferase activity requires active site lysine autoacetylation
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F. Bradley Johnson, Hestia Mellert, Rocco Perry, David W. Speicher, Jacques Côté, Kaye Speicher, Y. George Zheng, Santosh Hodawadekar, Rolf Sternglanz, Jiang Wu, Weiwei Dang, Nebiyu Abshiru, Madhusudan Srinivasan, Alain Verreault, Ronen Marmorstein, Emily Chen Ding, Dorine Rossetto, Jamel Johnson, Shelley L. Berger, Pierre Thibault, Steven B. McMahon, Chao Yang, and Hua Yuan
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Histone Acetyltransferases ,Lysine Acetyltransferases ,General Immunology and Microbiology ,General Neuroscience ,Lysine ,Acetyltransferases ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Histone ,Biochemistry ,Acetylation ,biology.protein ,Binding site ,KAT5 ,Molecular Biology - Abstract
The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.
- Published
- 2011
11. Structure of the Rtt109-AcCoA/Vps75 Complex and Implications for Chaperone-Mediated Histone Acetylation
- Author
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Katrina Meeth, Marc A. Holbert, Paul Drogaris, Alain Verreault, Ronen Marmorstein, Pierre Thibault, Hugo Wurtele, Yong Tang, Hua Yuan, Neda Delgoshaie, Benoit Guillemette, Eun Hye Lee, Philip A. Cole, and Chantal Durette
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Models, Molecular ,Saccharomyces cerevisiae Proteins ,Nucleosome assembly ,Recombinant Fusion Proteins ,Saccharomyces cerevisiae ,Gene Expression ,Cell Cycle Proteins ,Plasma protein binding ,Crystallography, X-Ray ,Genomic Instability ,Article ,Substrate Specificity ,Histones ,03 medical and health sciences ,Histone H3 ,0302 clinical medicine ,Acetyl Coenzyme A ,Structural Biology ,Humans ,Molecular Biology ,Histone Acetyltransferases ,030304 developmental biology ,0303 health sciences ,biology ,Lysine ,Acetylation ,Chromatin Assembly and Disassembly ,biology.organism_classification ,3. Good health ,Cell biology ,Histone ,Biochemistry ,Chaperone (protein) ,Mutagenesis, Site-Directed ,biology.protein ,030217 neurology & neurosurgery ,Molecular Chaperones ,Protein Binding - Abstract
SummaryYeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27, and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ∼12 Å diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3-H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones before deposition.
- Published
- 2011
12. Phosphorylated Rad18 directs DNA Polymerase η to sites of stalled replication
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Cyrus Vaziri, Satoshi Tateishi, Ying Zou, Naoko Kakusho, Hisao Masai, Alain Verreault, Laura R. Barkley, Tovah A. Day, and Komariah Palle
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DNA Replication ,DNA polymerase II ,Ubiquitin-Protein Ligases ,Eukaryotic DNA replication ,Cell Cycle Proteins ,DNA-Directed DNA Polymerase ,Protein Serine-Threonine Kinases ,Transfection ,DNA polymerase delta ,Article ,S Phase ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Proliferating Cell Nuclear Antigen ,Humans ,Phosphorylation ,Replication protein A ,S phase ,Research Articles ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,DNA clamp ,biology ,DNA replication ,Cell Biology ,Molecular biology ,3. Good health ,Cell biology ,DNA-Binding Proteins ,Microscopy, Fluorescence ,030220 oncology & carcinogenesis ,biology.protein ,Origin recognition complex - Abstract
Cdc7 phosphorylates Rad18 to integrate S phase progression with postreplication DNA repair, ensuring genome stability., The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability.
- Published
- 2010
13. Two Fundamentally Distinct PCNA Interaction Peptides Contribute to Chromatin Assembly Factor 1 Function
- Author
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Alain Verreault, Jack A. Kornblatt, Tom Rolef Ben-Shahar, Katherine L. B. Borden, Araceli G. Castillo, and Michael J. Osborne
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DNA Replication ,Models, Molecular ,Nucleosome assembly ,Molecular Sequence Data ,Eukaryotic DNA replication ,DNA polymerase delta ,Cell Line ,DNA replication factor CDT1 ,Mice ,Replication factor C ,Control of chromosome duplication ,Heterochromatin ,Proliferating Cell Nuclear Antigen ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,biology ,DNA replication ,Articles ,Cell Biology ,Molecular biology ,Nucleosomes ,Protein Structure, Tertiary ,Cell biology ,Chromatin Assembly Factor-1 ,Protein Subunits ,Mutagenesis, Site-Directed ,biology.protein ,Origin recognition complex ,lipids (amino acids, peptides, and proteins) ,Rabbits ,Peptides ,Sequence Alignment ,Transcription Factors - Abstract
Chromatin assembly factor 1 (CAF-1) deposits histones H3 and H4 rapidly behind replication forks through an interaction with the proliferating cell nuclear antigen (PCNA), a DNA polymerase processivity factor that also binds to a number of replication enzymes and other proteins that act on nascent DNA. The mechanisms that enable CAF-1 and other PCNA-binding proteins to function harmoniously at the replication fork are poorly understood. Here we report that the large subunit of human CAF-1 (p150) contains two distinct PCNA interaction peptides (PIPs). The N-terminal PIP binds strongly to PCNA in vitro but, surprisingly, is dispensable for nucleosome assembly and only makes a modest contribution to targeting p150 to DNA replication foci in vivo. In contrast, the internal PIP (PIP2) lacks one of the highly conserved residues of canonical PIPs and binds weakly to PCNA. Surprisingly, PIP2 is essential for nucleosome assembly during DNA replication in vitro and plays a major role in targeting p150 to sites of DNA replication. Unlike canonical PIPs, such as that of p21, the two p150 PIPs are capable of preferentially inhibiting nucleosome assembly, rather than DNA synthesis, suggesting that intrinsic features of these peptides are part of the mechanism that enables CAF-1 to function behind replication forks without interfering with other PCNA-mediated processes.
- Published
- 2009
14. Histone acetylation floods the human cell cycle and DNA damage response
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Alain Verreault, Neda Delgoshaie, and Hugo Wurtele
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Histone ,biology ,DNA damage ,Acetylation ,biology.protein ,Cell Biology ,Human cell ,Cell cycle ,Molecular Biology ,Developmental Biology ,Cell biology - Abstract
(2009). Histone acetylation floods the human cell cycle and DNA damage response. Cell Cycle: Vol. 8, No. 12, pp. 1818-1822.
- Published
- 2009
15. Artifactual Sulfation of Silver-stained Proteins
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Mathieu Courcelles, Alain Verreault, Sylvain Meloche, Maria Marcantonio, Pierre Thibault, Marlene Gharib, and Sylvia G. Lehmann
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Gel electrophoresis ,Thiosulfate ,Chemistry ,Biochemistry ,Analytical Chemistry ,Staining ,Silver stain ,chemistry.chemical_compound ,Sulfation ,Phosphorylation ,Threonine ,Tyrosine ,Molecular Biology - Abstract
Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues. These two post-translational modifications are often difficult to distinguish because of their similar MS fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine, and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyzes the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and Escherichia coli cell extracts indicated that several serine, threonine, and tyrosine residues were sulfated using silver staining protocols but not following Coomassie Blue staining. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction, and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.
- Published
- 2009
16. Structural Basis for the Recognition of Histone H4 by the Histone-Chaperone RbAp46
- Author
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Wei Zhang, Ernest D. Laue, Natalia V. Murzina, Tom Rolef Ben-Shahar, Alain Verreault, Stephen H. McLaughlin, J. Venkatesh Pratap, Jose Vicente-Garcia, Xue-Yuan Pei, Mike Sparkes, and Ben F. Luisi
- Subjects
Models, Molecular ,PROTEINS ,Molecular Sequence Data ,Biology ,Article ,Histone H4 ,Histones ,03 medical and health sciences ,Histone H3 ,0302 clinical medicine ,Histone H1 ,Structural Biology ,Histone H2A ,Histone methylation ,Histone code ,Humans ,Histone octamer ,Amino Acid Sequence ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Sequence Homology, Amino Acid ,Nuclear Proteins ,DNA ,Cell biology ,Biochemistry ,Histone methyltransferase ,Retinoblastoma-Binding Protein 7 ,Carrier Proteins ,030217 neurology & neurosurgery ,Molecular Chaperones ,Protein Binding - Abstract
Summary RbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed β propeller structure and binds histone H4 in a groove formed between an N-terminal α helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.
- Published
- 2008
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17. Regulation of Histone H3 Lysine 56 Acetylation in Schizosaccharomyces pombe
- Author
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Hiroshi Masumoto, Stephen P. Jackson, Alain Verreault, Kyle M. Miller, Blerta Xhemalce, Tony Kouzarides, Benoit Arcangioli, and Robert Driscoll
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DNA Replication ,G2 Phase ,Saccharomyces cerevisiae Proteins ,SAP30 ,Biochemistry ,S Phase ,Histones ,Histone H3 ,Histone H1 ,Schizosaccharomyces ,Histone H2A ,Histone code ,DNA Breaks, Double-Stranded ,Histone octamer ,Molecular Biology ,Histone Acetyltransferases ,Sequence Homology, Amino Acid ,biology ,Lysine ,Acetylation ,Cell Biology ,Histone acetyltransferase ,biology.protein ,Schizosaccharomyces pombe Proteins ,Histone deacetylase ,Protein Processing, Post-Translational ,Mutagens - Abstract
In Saccharomyces cerevisiae, acetylation of lysine 56 (Lys-56) in the globular domain of histone H3 plays an important role in response to genotoxic agents that interfere with DNA replication. However, the regulation and biological function of this modification are poorly defined in other eukaryotes. Here we show that Lys-56 acetylation in Schizosaccharomyces pombe occurs transiently during passage through S-phase and is normally removed in G(2). Genotoxic agents that cause DNA double strand breaks during replication elicit a delay in deacetylation of histone H3 Lys-56. In addition, mutant cells that cannot acetylate Lys-56 are acutely sensitive to genotoxic agents that block DNA replication. Moreover, we show that Spbc342.06cp, a previously uncharacterized open reading frame, encodes the functional homolog of S. cerevisiae Rtt109, and that this protein acetylates H3 Lys-56 both in vitro and in vivo. Altogether, our results indicate that both the regulation of histone H3 Lys-56 acetylation by its histone acetyltransferase and histone deacetylase and its role in the DNA damage response are conserved among two distantly related yeast model organisms.
- Published
- 2007
18. Histone H3 Lysine 56 Acetylation: A New Twist in the Chromosome Cycle
- Author
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Colin Logie, Anil Ozdemir, Alain Verreault, Paul Fitzjohn, and Hiroshi Masumoto
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DNA Replication ,Models, Molecular ,Saccharomyces cerevisiae Proteins ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Biology ,SAP30 ,Models, Biological ,Chromosomes ,Histones ,Histone H3 ,Acetyltransferases ,Histone code ,Nucleosome ,Histone octamer ,Molecular Biology ,Lysine ,Cell Cycle ,Acetylation ,Cell Biology ,Molecular biology ,Cell biology ,Histone ,biology.protein ,Histone deacetylase ,DNA Damage ,Molecular Chaperones ,Developmental Biology - Abstract
Several recent reports have identified lysine 56 (K56) as a novel site of acetylation in yeast histone H3. K56 acetylation is predicted to disrupt some of the histone-DNA interactions at the entry and exit points of the nucleosome core particle. This modification occurs in virtually all the newly synthesised histones that are deposited into chromatin during S-phase. Cells with mutations that block K56 acetylation show increased genome instability and hypersensitivity to genotoxic agents that interfere with replication. Removal of K56 acetylation takes place in the G2/M phase of the cell cycle and is dependent upon Hst3 and Hst4, two proteins that are related to the NAD+-dependent histone deacetylase Sir2. In response to DNA damage checkpoint activation during S-phase, expression of Hst3/Hst4 is delayed to extend the window of opportunity in which K56 acetylation can act in the DNA damage response. The high abundance of histone H3 K56 acetylation, its regulation and strategic location in the nucleosome core particle raise a number of fascinating issues that we discuss here.
- Published
- 2006
19. Transcriptional Coactivator PC4, a Chromatin-Associated Protein, Induces Chromatin Condensation
- Author
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Kohji Hizume, Kiran Batta, Chandrima Das, Stephanie Lorain, Tapas K. Kundu, Parag P. Sadhale, Alain Verreault, B. R. Prashanth Kumar, Semanti Ganguly, Shrikanth S. Gadad, and Kunio Takeyasu
- Subjects
Histone-modifying enzymes ,Transcription, Genetic ,Biology ,Microscopy, Atomic Force ,Chromosomes ,Chromatin remodeling ,Histones ,Histone H1 ,Humans ,RNA, Small Interfering ,Molecular Biology ,Metaphase ,ChIA-PET ,Oligonucleotide Array Sequence Analysis ,Microbiology & Cell Biology ,Cell Cycle ,Articles ,Cell Biology ,ChIP-on-chip ,Chromatin Assembly and Disassembly ,Chromatin ,Cell biology ,ChIP-sequencing ,DNA-Binding Proteins ,HeLa Cells ,Transcription Factors ,Bivalent chromatin - Abstract
Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G2/M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization.
- Published
- 2006
20. Histone H3 Lysine 4 Mono-methylation does not Require Ubiquitination of Histone H2B
- Author
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Ramon Sendra, Daniéle Moinier, Mercè Pamblanco, Pierre Luciano, Pierre-Marie Dehé, Alain Verreault, Vincent Géli, Régine Lebrun, and Vicente Tordera
- Subjects
Histone H3 Lysine 4 ,Ubiquitin ,Lysine ,Saccharomyces cerevisiae ,Biology ,Methylation ,environment and public health ,Molecular biology ,Cell biology ,Histones ,Histone H1 ,Structural Biology ,Histone methyltransferase ,Histone H2A ,Histone methylation ,Histone H2B ,Histone code ,Histone octamer ,Molecular Biology - Abstract
The yeast Set1-complex catalyzes histone H3 lysine 4 (H3K4) methylation. Using N-terminal Edman sequencing, we determined that 50% of H3K4 is methylated and consists of roughly equal amounts of mono, di and tri-methylated H3K4. We further show that loss of either Paf1 of the Paf1 elongation complex, or ubiquitination of histone H2B, has only a modest effect on bulk histone mono-methylation at H3K4. Despite the fact that Set1 recruitment decreases in paf1delta cells, loss of Paf1 results in an increase of H3K4 mono-methylation at the 5' coding region of active genes, suggesting a Paf1-independent targeting of Set1. In contrast to Paf1 inactivation, deleting RTF1 affects H3K4 mono-methylation at the 3' coding region of active genes and results in a decrease of global H3K4 mono-methylation. Our results indicate that the requirements for mono-methylation are distinct from those for H3K4 di and tri-methylation, and point to differences among members of the Paf1 complex in the regulation of H3K4 methylation.
- Published
- 2005
21. Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin
- Author
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Mitsuru Okuwaki, Debbie Lyon, Alain Verreault, Helen R. Mott, Daniel Nietlispach, Miriam Hirshberg, Natalia V. Murzina, Ernest D. Laue, Abarna Thiru, and Peter R. Nielsen
- Subjects
Chromosomal Proteins, Non-Histone ,Heterochromatin ,Amino Acid Motifs ,Molecular Sequence Data ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Histones ,Mice ,Histone H3 ,Protein structure ,Animals ,Humans ,Histone code ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Peptide sequence ,Chromo shadow domain ,Genetics ,General Immunology and Microbiology ,General Neuroscience ,Protein Structure, Tertiary ,Chromatin ,Cell biology ,Chromobox Protein Homolog 5 ,Heterochromatin protein 1 ,Protein Binding - Abstract
HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1beta bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a beta-sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif-containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1beta to heterochromatin requires shadow domain interactions with PXVXL-containing proteins in addition to chromo domain recognition of Lys-9-methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys-9-methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.
- Published
- 2004
22. DNA base excision repair of uracil residues in reconstituted nucleosome core particles
- Author
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Alain Verreault, Tomas Lindahl, and Hilde Nilsen
- Subjects
DNA Repair ,DNA repair ,DNA, Ribosomal ,General Biochemistry, Genetics and Molecular Biology ,AP endonuclease ,Animals ,AP site ,Uracil ,Molecular Biology ,DNA Primers ,Base Sequence ,General Immunology and Microbiology ,biology ,General Neuroscience ,RNA, Ribosomal, 5S ,Articles ,Base excision repair ,Linker DNA ,Nucleosomes ,Very short patch repair ,Biochemistry ,DNA glycosylase ,biology.protein ,Biophysics ,Echinodermata ,Nucleotide excision repair - Abstract
The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.
- Published
- 2002
23. Heterochromatin Dynamics in Mouse Cells
- Author
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Natalia V. Murzina, Bruce Stillman, Ernest D. Laue, and Alain Verreault
- Subjects
endocrine system ,animal structures ,Nucleosome assembly ,Cell Biology ,Solenoid (DNA) ,Biology ,Chromatin ,Cell biology ,Non-histone protein ,embryonic structures ,Histone code ,Nucleosome ,Heterochromatin protein 1 ,Chromatin Assembly Factor-1 ,Molecular Biology - Abstract
Mechanisms contributing to the maintenance of heterochromatin in proliferating cells are poorly understood. We demonstrate that chromatin assembly factor 1 (CAF-1) binds to mouse HP1 proteins via an N-terminal domain of its p150 subunit, a domain dispensable for nucleosome assembly during DNA replication. Mutations in p150 prevent association with HP1 in heterochromatin in cells that are not in S phase and the formation of CAF-1-HP1 complexes in nascent chromatin during DNA replication in vitro. We suggest that CAF-1 p150 has a heterochromatin-specific function distinct from its nucleosome assembly function during S phase. Just before mitosis, CAF-1 p150 and some HP1 progressively dissociate from heterochromatin concomitant with histone H3 phosphorylation. The HP1 proteins reassociate with chromatin at the end of mitosis, as histone H3 is dephosphorylated.
- Published
- 1999
24. Nucleosome Assembly by a Complex of CAF-1 and Acetylated Histones H3/H4
- Author
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Paul D. Kaufman, Bruce Stillman, Alain Verreault, and Ryuji Kobayashi
- Subjects
Cell Extracts ,DNA Replication ,Cytoplasm ,DNA, Complementary ,Saccharomyces cerevisiae Proteins ,Nucleosome assembly ,Chromosomal Proteins, Non-Histone ,Molecular Sequence Data ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Histones ,Histone H4 ,Histone H1 ,Acetyltransferases ,Histone H2A ,Humans ,Histone code ,Amino Acid Sequence ,Histone octamer ,Chromatin Assembly Factor-1 ,Cloning, Molecular ,Histone Acetyltransferases ,Cell Nucleus ,Biochemistry, Genetics and Molecular Biology(all) ,Lysine ,Chromatin Assembly Factor I ,Acetylation ,Molecular biology ,Chromatin ,Nucleosomes ,Cell biology ,DNA-Binding Proteins ,Molecular Weight ,Transcription Factors - Abstract
Chromatin assembly factor 1 (CAF-1) assembles nucleosomes in a replication-dependent manner. The small subunit of CAF-1 (p48) is a member of a highly conserved subfamily of WD-repeat proteins. There are at least two members of this subfamily in both human (p46 and p48) and yeast cells (Hat2p, a subunit of the B-type H4 acetyltransferase, and Msi1p). Human p48 can bind to histone H4 in the absence of CAF-1 p150 and p60. p48, also a known subunit of a histone deacetylase, copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150, p60, p48) and H3 and H4, and promotes DNA replication-dependent chromatin assembly. CAC histone H4 exhibits a novel pattern of lysine acetylation that overlaps with, but is distinct from, that reported for newly synthesized H4 isolated from nascent chromatin. Our data suggest that CAC is a key intermediate of the de novo nucleosome assembly pathway and that the p48 subunit participates in other aspects of histone metabolism.
- Published
- 1996
25. Modulation of Heterochromatin Protein 1 Dynamics in Primary Mammalian Cells
- Author
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Alain Verreault, Belaïd Sekkali, Kyoko Hiragami, Stamatis N. Pagakis, Dimitris Kioussis, Debbie Lyon, and Richard Festenstein
- Subjects
Euchromatin ,Chromosomal Proteins, Non-Histone ,Heterochromatin ,T-Lymphocytes ,Biology ,Lymphocyte Activation ,Methylation ,Fluorescence ,Chromatin remodeling ,Histones ,Mice ,Non-histone protein ,Animals ,Transcription factor ,Cells, Cultured ,Binding Sites ,Microscopy, Confocal ,Multidisciplinary ,Fluorescence recovery after photobleaching ,Molecular biology ,Chromatin ,Cell biology ,Kinetics ,Chromobox Protein Homolog 5 ,Heterochromatin protein 1 ,Dimerization ,Fluorescence Recovery After Photobleaching - Abstract
Heterochromatin protein 1 (HP1β), a key component of condensed DNA, is strongly implicated in gene silencing and centromeric cohesion. Heterochromatin has been considered a static structure, stabilizing crucial aspects of nuclear organization and prohibiting access to transcription factors. We demonstrate here, by fluorescence recovery after photobleaching, that a green fluorescent protein–HP1β fusion protein is highly mobile within both the euchromatin and heterochromatin of ex vivo resting murine T cells. Moreover, T cell activation greatly increased this mobility, indicating that such a process may facilitate (hetero)chromatin remodeling and permit access of epigenetic modifiers and transcription factors to the many genes that are consequently derepressed.
- Published
- 2003
26. Histone Deacetylase Inhibitors Globally Enhance H3/H4 Tail Acetylation Without Affecting H3 Lysine 56 Acetylation
- Author
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Eun Hye Lee, Pierre Thibault, Eric Bonneil, Véronique Bourdeau, Gerardo Ferbeyre, Alain Verreault, Christelle Pomiès, Valérie Villeneuve, and Paul Drogaris
- Subjects
0303 health sciences ,Histone deacetylase 5 ,Multidisciplinary ,HDAC11 ,Histone deacetylase 2 ,SAP30 ,Biology ,HDAC4 ,Molecular biology ,Article ,3. Good health ,Cell biology ,03 medical and health sciences ,Histone H3 ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Histone H2A ,Histone deacetylase ,030304 developmental biology - Abstract
Histone deacetylase inhibitors (HDACi) represent a promising avenue for cancer therapy. We applied mass spectrometry (MS) to determine the impact of clinically relevant HDACi on global levels of histone acetylation. Intact histone profiling revealed that the HDACi SAHA and MS-275 globally increased histone H3 and H4 acetylation in both normal diploid fibroblasts and transformed human cells. Histone H3 lysine 56 acetylation (H3K56ac) recently elicited much interest and controversy due to its potential as a diagnostic and prognostic marker for a broad diversity of cancers. Using quantitative MS, we demonstrate that H3K56ac is much less abundant than previously reported in human cells. Unexpectedly, in contrast to H3/H4 N-terminal tail acetylation, H3K56ac did not increase in response to inhibitors of each class of HDACs. In addition, we demonstrate that antibodies raised against H3K56ac peptides cross-react against H3 N-terminal tail acetylation sites that carry sequence similarity to residues flanking H3K56.
- Published
- 2012
27. Histone H3 Lysine 56 Acetylation and the Response to DNA Replication Fork Damage
- Author
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Jonas F. Dorn, Edlie St-Hilaire, Hugo Wurtele, Philippe Pasero, Julien Bacal, Gitte Schalck Kaiser, Michael Lisby, Paul S. Maddox, Eun Hye Lee, Alain Verreault, Sarah Tsao, Institut de Recherche en Immunologie et en Cancérologie [UdeM-Montréal] (IRIC), Université de Montréal (UdeM), Institut de génétique humaine (IGH), and Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
DNA Replication ,Saccharomyces cerevisiae Proteins ,DNA Repair ,DNA repair ,DNA damage ,Eukaryotic DNA replication ,Antineoplastic Agents ,Saccharomyces cerevisiae ,Biology ,Histones ,03 medical and health sciences ,0302 clinical medicine ,Control of chromosome duplication ,Hydroxyurea ,DNA, Fungal ,Molecular Biology ,Replication protein A ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,[SDV.GEN]Life Sciences [q-bio]/Genetics ,Lysine ,DNA replication ,Acetylation ,Cell Biology ,Articles ,DNA Replication Fork ,Methyl Methanesulfonate ,Molecular biology ,3. Good health ,Chromatin ,030220 oncology & carcinogenesis ,Mutation ,Camptothecin ,DNA Damage ,Mutagens - Abstract
In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.
- Published
- 2012
28. LMO2 Regulates DNA Replication in Hematopoietic Cells
- Author
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EL Bachir Affar, Benoit Grondin, Véronique Lisi, Trang Hoang, Marie-Claude Sincennes, Magali Humbert, Christophe Cazaux, Alain Verreault, and Nazar Mashtalir
- Subjects
DNA re-replication ,biology ,Immunology ,DNA replication ,Eukaryotic DNA replication ,Cell Biology ,Hematology ,Pre-replication complex ,Biochemistry ,Molecular biology ,DNA replication factor CDT1 ,Licensing factor ,Control of chromosome duplication ,hemic and lymphatic diseases ,biology.protein ,Origin recognition complex - Abstract
Oncogenic transcription factors are major drivers in acute leukemias. These oncogenes are believed to subvert normal cell identity via the establishment of gene expression programs that dictate cell differentiation and growth. The LMO2 oncogene, which is commonly activated in T-cell acute lymphoblastic leukemia (T-ALL), has a well-established function in transcription regulation. We and others previously demonstrated that LMO1 or LMO2 collaborate with the SCL transcription factor to activate a self-renewal program that converts non self-renewing progenitors into pre-leukemic stem cells. Here we demonstrate a non-transcriptional role of LMO2 in controlling cell fate by directly promoting DNA replication, a hitherto unrecognized mechanism that might also account for its oncogenic properties. To address the question whether LMO2 controls other functions via protein-protein interactions, we performed a proteome-wide screen for LMO2 interaction partners in Kit+ Lin- cells. In addition to known LMO2-interacting proteins such as LDB1 and to proteins associated with transcription, we unexpectedly identified new interactions with three essential DNA replication enzymes, namely minichromosome 6 (MCM6), DNA polymerase delta (POLD1) and DNA primase (PRIM1). First, we show that in Kit+ hematopoietic cells (TF-1), all components of the pre-replication complex co-immunoprecipitate with LMO2 but not with SCL, suggesting a novel SCL-independent function. Second, LMO2 is recruited to DNA replication origins in these cells together with MCM5. Third, tethering LMO2 to synthetic DNA sequences is sufficient to transform these into origins of replication. Indeed, we show by DNA capture that LMO2 fused to the DNA binding domain of GAL4 is sufficient to recruit DNA replication proteins to GAL4 binding sites on DNA. In vivo, this recruitment is sufficient to drive DNA replication in a manner which is dependent on the integrity of the GAL4 binding sites. These results provide unambiguous evidence for a role of LMO2 in directly controlling DNA replication. Cell cycle and cell differentiation are tightly coordinated during normal hematopoiesis, both during erythroid differentiation and during thymocyte development. We next addressed the functional importance of LMO2 in these two lineages. Erythroid cell differentiation proceeds through different stages from the CD71+Ter119- to the CD71-Ter119+. These stages are also distinguishable by morphological criteria. We observe that LMO2 protein levels directly correlate with the proportion of cells in S phase, i.e. both LMO2 levels and the proportions of cycling cells decrease with terminal erythroid differentiation. Strikingly, lowering LMO2 levels in fetal liver erythroid progenitors via shRNAs decreases the proportion of cells in S phase and arrests Epo-dependent cell growth. Despite a drastic decrease in the numbers of erythroid precursors, these cells differentiate readily to the CD71-Ter119+ stage. Therefore, LMO2 levels dictate cell fate in the erythroid lineage, by favoring DNA replication at the expense of terminal maturation. Conversely, ectopic expression in thymocytes induces DNA replication and drives cells into cell cycle, causing differentiation blockade. Our results define a novel role for the oncogenic transcription factor LMO2 in directly promoting DNA synthesis. To our knowledge, this is the first evidence for a non-transcriptional function of the LMO2 oncogene that drives cell cycle at the expense of differentiation, favouring progenitor cell expansion in the thymus, and causing T-ALL when ectopically expressed in the T lineage. We propose that the non-transcriptional control of DNA replication uncovered here for LMO2 may be a more common function of oncogenic transcription factors than previously appreciated. Disclosures No relevant conflicts of interest to declare.
- Published
- 2015
29. H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation
- Author
-
Hsiu-Hsu Sophia Lin, Pierre Thibault, Eric Bonneil, Kyoko Hiragami-Hamada, Alain Verreault, Richard Festenstein, Axel Imhof, Harry Armstrong, Paul Drogaris, and Benoit Guillemette
- Subjects
Cancer Research ,Histone H3 Lysine 4 ,Saccharomyces cerevisiae Proteins ,lcsh:QH426-470 ,Saccharomyces cerevisiae ,Biology ,Molecular Biology/Histone Modification ,environment and public health ,Methylation ,Gene Expression Regulation, Enzymologic ,Euchromatin ,Histones ,03 medical and health sciences ,0302 clinical medicine ,Sirtuin 2 ,Histone H1 ,Heterochromatin ,Histone H2A ,Histone methylation ,Genetics ,Histone code ,Gene Regulatory Networks ,Molecular Biology/Chromatin Structure ,Promoter Regions, Genetic ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,Silent Information Regulator Proteins, Saccharomyces cerevisiae ,030304 developmental biology ,Histone Acetyltransferases ,0303 health sciences ,Lysine ,Acetylation ,Histone-Lysine N-Methyltransferase ,Molecular Biology/Transcription Initiation and Activation ,lcsh:Genetics ,Biochemistry ,Histone methyltransferase ,Histone deacetylase ,Chromatin immunoprecipitation ,030217 neurology & neurosurgery ,Research Article - Abstract
Methylation of histone H3 lysine 4 (H3K4me) is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac) has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs) Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC) Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP), we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3), a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS), which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me)., Author Summary In the nucleus of mammals and yeast, DNA is packaged by forming complexes with histone proteins in a structure called the nucleosome, the basic building block of chromatin. The tails of the histones protrude from the nucleosome and can be marked on many amino acid residues by chemical modifications such as methylation and acetylation. A highly studied modification, which is robustly associated with active gene promoters, is histone H3 lysine 4 methylation. We describe here a novel modification, histone H3 lysine 4 acetylation (H3K4ac), which can occur on the same lysine of the histone H3 tail (but not at the same time as methylation). We have identified the enzymes responsible for depositing and removing this mark and mapped its distribution throughout the yeast genome. We found that H3K4ac is present on active genes and is important for the full expression of a subset of them. Strikingly, H3K4 methylation was found in the same promoters as H3K4ac and contributes to regulate the abundance and localisation of H3K4ac. This example of cross-talk between two different modifications of the same residue has fundamental implications for understanding how genes are activated and how their packaging in the nucleus controls this process.
- Published
- 2011
30. Regulation of the DNA damage response and gene expression by the Dot1L histone methyltransferase and the 53Bp1 tumour suppressor
- Author
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Noel F. Lowndes, Muriel Grenon, Nebiyu Abshiru, Jennifer FitzGerald, Enda O'Connell, Paul Drogaris, Sylvie Moureau, Pierre Thibault, and Alain Verreault
- Subjects
Chromosomal Proteins, Non-Histone ,mammalian-cells ,Histones ,Mice ,DNA Breaks, Double-Stranded ,Regulation of gene expression ,0303 health sciences ,double-strand breaks ,Multidisciplinary ,biology ,030302 biochemistry & molecular biology ,Genetics and Genomics/Gene Expression ,Chromatin ,3. Good health ,DNA-Binding Proteins ,Genetics and Genomics/Chromosome Biology ,Histone ,h3 lysine-79 methyltransferase ,Histone methyltransferase ,Gene Knockdown Techniques ,DNA methylation ,set domain ,Histone Methyltransferases ,saccharomyces-cerevisiae ,Medicine ,Cell Biology/Nuclear Structure and Function ,Tumor Suppressor p53-Binding Protein 1 ,DOT1L Gene ,nucleosome core ,Signal Transduction ,Research Article ,DNA repair ,DNA damage ,Science ,Molecular Biology/Histone Modification ,Cell Line ,03 medical and health sciences ,Genetics and Genomics/Epigenetics ,Animals ,silencing protein ,Molecular Biology/Chromatin Structure ,Cell Biology/Gene Expression ,030304 developmental biology ,Molecular Biology/Recombination ,Molecular Biology/DNA Repair ,Gene Expression Profiling ,Lysine ,Tumor Suppressor Proteins ,Histone-Lysine N-Methyltransferase ,Methyltransferases ,Microarray Analysis ,Molecular biology ,enac-alpha ,Gene Expression Regulation ,biology.protein ,methylation ,DNA Damage - Abstract
BackgroundDot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.Methodology/principal findingsUsing the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.Conclusions/significanceTaken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.
- Published
- 2010
31. Modulation of histone H3 lysine 56 acetylation as an antifungal therapeutic strategy
- Author
-
Martine Raymond, Paul Drogaris, Alaka Mullick, Guylaine Lépine, Eun Hye Lee, Alain Verreault, Hugo Wurtele, Pierre Thibault, Sarah Tsao, and Jessy Tremblay
- Subjects
yeast ,Histones ,immunology ,Drug Delivery Systems ,inhibitors ,saccharomyces cerevisiae ,Candida albicans ,Histone Acetyltransferases ,Fungal protein ,biology ,diffusion ,Acetylation ,in vitro ,General Medicine ,vitro ,Corpus albicans ,nucleic acids ,Histone ,Acetyltransferase ,candida ,acid ,antifungal agents ,cell cycle ,epidemiology ,biotechnology ,Niacinamide ,skin ,mice ,Cell Survival ,mouse model ,enzymes ,DNA replication ,chemistry ,Histone Deacetylases ,General Biochemistry, Genetics and Molecular Biology ,Article ,Microbiology ,Fungal Proteins ,Histone H3 ,sirtuins ,Animals ,mammals ,gene ,genome ,therapy ,activity ,flow cytometry ,aging ,DNA ,cell ,biology.organism_classification ,candidiasis ,Molecular biology ,virulence ,gene expression ,biology.protein ,RNA ,candida albicans ,protein - Abstract
Candida albicans is a major fungal pathogen that causes serious systemic and mucosal infections in immunocompromised individuals. In yeast, histone H3 Lys56 acetylation (H3K56ac) is an abundant modification regulated by enzymes that have fungal-specific properties, making them appealing targets for antifungal therapy. Here we demonstrate that H3K56ac in C. albicans is regulated by the RTT109 and HST3 genes, which respectively encode the H3K56 acetyltransferase (Rtt109p) and deacetylase (Hst3p). We show that reduced levels of H3K56ac sensitize C. albicans to genotoxic and antifungal agents. Inhibition of Hst3p activity by conditional gene repression or nicotinamide treatment results in a loss of cell viability associated with abnormal filamentous growth, histone degradation and gross aberrations in DNA staining. We show that genetic or pharmacological alterations in H3K56ac levels reduce virulence in a mouse model of C. albicans infection. Our results demonstrate that modulation of H3K56ac is a unique strategy for treatment of C. albicans and, possibly, other fungal infections. -¬ 2010 Nature America, Inc. All rights reserved
- Published
- 2010
32. Fungal Rtt109 histone acetyltransferase is an unexpected structural homolog of metazoan p300/CBP
- Author
-
Pierre Thibault, Marlene Gharib, Yong Tang, Alain Verreault, Walter Rocha, Philip A. Cole, Eva Jiang, Katrina Meeth, Marc A. Holbert, Hugo Wurtele, and Ronen Marmorstein
- Subjects
Models, Molecular ,Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Crystallography, X-Ray ,Protein Structure, Secondary ,Article ,Histones ,03 medical and health sciences ,Histone H3 ,Structure-Activity Relationship ,Histone H1 ,Structural Biology ,Acetyl Coenzyme A ,Histone H2A ,parasitic diseases ,Histone code ,Animals ,p300-CBP Transcription Factors ,Histone octamer ,Molecular Biology ,030304 developmental biology ,Histone Acetyltransferases ,0303 health sciences ,Binding Sites ,biology ,Lysine ,030302 biochemistry & molecular biology ,Acetylation ,Histone acetyltransferase ,3. Good health ,Biochemistry ,Mutagenesis ,Structural Homology, Protein ,Histone methyltransferase ,biology.protein ,Mutant Proteins ,Mutagens - Abstract
Rtt109, also known as KAT11, is a recently characterized fungal-specific histone acetyltransferase (HAT) that modifies histone H3 lysine 56 (H3K56) to promote genome stability. Rtt109 does not show sequence conservation with other known HATs and depends on association with either of two histone chaperones, Asf1 or Vps75, for HAT activity. Here we report the X-ray crystal structure of an Rtt109-acetyl coenzyme A complex and carry out structure-based mutagenesis, combined with in vitro biochemical studies of the Rtt109-Vps75 complex and studies of Rtt109 function in vivo. The Rtt109 structure reveals noteworthy homology to the metazoan p300/CBP HAT domain but exhibits functional divergence, including atypical catalytic properties and mode of cofactor regulation. The structure reveals a buried autoacetylated lysine residue that we show is also acetylated in the Rtt109 protein purified from yeast cells. Implications for understanding histone substrate and chaperone binding by Rtt109 are discussed.
- Published
- 2008
33. Clothing up DNA for all seasons: Histone chaperones and nucleosome assembly pathways
- Author
-
Alain Verreault and Walter Rocha
- Subjects
DNA Replication ,Nucleosome assembly ,Transcription, Genetic ,Molecular Sequence Data ,Biophysics ,Embryonic Development ,Biochemistry ,Chromatin remodeling ,Histones ,03 medical and health sciences ,Mice ,Chromatin assembly ,0302 clinical medicine ,Control of chromosome duplication ,Structural Biology ,Genetics ,Histone chaperone ,Histone code ,Animals ,Humans ,Chromatin Assembly Factor-1 ,Amino Acid Sequence ,Gene Silencing ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,DNA replication ,Acetylation ,Cell Biology ,DNA ,Chromatin ,Cell biology ,Nucleosomes ,Histone ,biology.protein ,030217 neurology & neurosurgery ,Molecular Chaperones - Abstract
In eukaryotes, the packaging of DNA into chromatin is essential for cell viability. Several important DNA metabolic events require the transient disruption of chromatin structure, but cells have evolved a number of elaborate pathways that operate throughout the cell cycle to prevent the deleterious effects of chromatin erosion. In this review, we describe a number of distinct nucleosome assembly pathways that function during DNA replication, transcription, cellular senescence and early embryogenesis. In addition, we illustrate some of the physiological consequences associated with defects in nucleosome assembly pathways.
- Published
- 2008
34. Histone post-translational modifications and the response to DNA double-strand breaks
- Author
-
Alain Verreault and Hugo Wurtele
- Subjects
Histone-modifying enzymes ,DNA Repair ,Acetylation ,Cell Cycle Proteins ,Cell Biology ,Biology ,Molecular biology ,Methylation ,Models, Biological ,Chromatin remodeling ,Cell biology ,Chromatin ,Nucleosomes ,Histones ,Histone ,Histone H2A ,biology.protein ,Histone code ,Nucleosome ,Animals ,Humans ,Phosphorylation ,Protein Processing, Post-Translational ,Epigenomics ,DNA Damage - Abstract
The packaging of DNA into chromatin creates a number of significant barriers to the detection of DNA lesions and their timely and accurate repair. Eukaryotic cells have evolved a number of enzymes that modulate chromatin structure and facilitate DNA repair. Recent research illustrates how nucleosome remodelling enzymes cooperate with both DNA-damage-inducible and constitutive histone modifications to promote many facets of the cellular response to DNA damage.
- Published
- 2006
35. Maintenance DNA methylation of nucleosome core particles
- Author
-
Mitsuru Okuwaki and Alain Verreault
- Subjects
Genetics ,DNA (Cytosine-5-)-Methyltransferase 1 ,DNA clamp ,DNA replication ,RNA, Ribosomal, 5S ,Cell Biology ,Biology ,DNA Methylation ,Biochemistry ,Cell biology ,Nucleosomes ,Epigenetics of physical exercise ,Histone ,embryonic structures ,DNA methylation ,Histone methylation ,biology.protein ,Nucleosome ,Animals ,DNA (Cytosine-5-)-Methyltransferases ,Molecular Biology ,Epigenomics - Abstract
The enzyme responsible for maintenance methylation of CpG dinucleotides in vertebrates is DNMT1. The presence of DNMT1 in DNA replication foci raises the issue of whether this enzyme needs to gain access to nascent DNA before its packaging into nucleosomes, which occurs very rapidly behind the replication fork. Using nucleosomes positioned along the 5 S rRNA gene, we find that DNMT1 is able to methylate a number of CpG sites even when the DNA major groove is oriented toward the histone surface. However, we also find that the ability of DNMT1 to methylate nucleosomal sites is highly dependent on the nature of the DNA substrate. Although nucleosomes containing the Air promoter are refractory to methylation irrespective of target cytosine location, nucleosomes reconstituted onto the H19 imprinting control region are more accessible. These results argue that although DNMT1 is intrinsically capable of methylating some DNA sequences even after their packaging into nucleosomes, this is not the case for at least a fraction of DNA sequences whose function is regulated by DNA methylation.
- Published
- 2003
36. LMO1/2 regulates DNA replication in hematopoietic cells
- Author
-
Benoit Grondin, Magali Humbert, Alain Verreault, Mathieu Tremblay, Marie-Claude Sincennes, André Haman, and Trang Hoang
- Subjects
DNA replication factor CDT1 ,Cancer Research ,Haematopoiesis ,biology ,Control of chromosome duplication ,Genetics ,DNA replication ,biology.protein ,Eukaryotic DNA replication ,Cell Biology ,Hematology ,Molecular Biology ,Cell biology - Published
- 2014
37. The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1- mediated nucleosome assembly onto replicated DNA in vitro
- Author
-
Alain Verreault, Kei-ichi Shibahara, and Bruce Stillman
- Subjects
DNA Replication ,Nucleosome assembly ,Chromosomal Proteins, Non-Histone ,Solenoid (DNA) ,Biology ,Histones ,Histone code ,Nucleosome ,Humans ,Chromatin Assembly Factor-1 ,Histone octamer ,Multidisciplinary ,Binding Sites ,Cell-Free System ,Models, Genetic ,Acetylation ,Biological Sciences ,Molecular biology ,Linker DNA ,Peptide Fragments ,Recombinant Proteins ,Cell biology ,Nucleosomes ,DNA-Binding Proteins ,Chromatosome ,Protein Binding - Abstract
An in vitro reconstitution system for the analysis of replication-coupled nucleosome assembly is described. In this “two-step system,” nucleosome assembly is performed in a separate reaction from DNA replication, wherein purified newly replicated DNA remains noncovalently marked for subsequent chromatin assembly factor-1 (CAF-1)-dependent nucleosome assembly. Because the nucleosome assembly is performed separately from the DNA replication step, this system is more versatile and biochemically tractable when compared with nucleosome assembly during simian virus 40 (SV40) DNA replication. The N-terminal domains of histones H3 and H4 play an important but redundant function in nucleosome assembly in the budding yeast, Saccharomyces cerevisiae . It had been proposed that at least one tail of histone H3 or H4 is required for replication-coupled nucleosome assembly. However, we demonstrate that the N-terminal domains of both histone H3 and H4 are dispensable for CAF-1-mediated formation of nucleosome cores onto newly replicated DNA in vitro . CAF-1 and each of its individual subunits stably bound to recombinant (H3.H4) 2 tetramers lacking the N-terminal domains of both H3 and H4. Therefore, the N-terminal tails of histone H3 and H4 that contain the specific acetylation sites are not necessary for CAF-1-dependent nucleosome assembly onto replicated DNA. We suggest that the histone acetylation may be required for a CAF-1 independent pathway or function after deposition, by marking of newly replicated chromatin.
- Published
- 2000
38. Methods for biochemical study of poly(ADP-ribose) metabolism in vitro and in vivo
- Author
-
Caroline Duchaine, Girish M. Shah, Alain Verreault, Gino Brochu, Jean Lagueux, James B. Kirkland, Serge Desnoyers, Jean-Christophe Hoflack, G.G. Poirier, and D. Poirier
- Subjects
Poly Adenosine Diphosphate Ribose ,DNA Repair ,Glycoside Hydrolases ,Blotting, Western ,Biophysics ,Apoptosis ,Biochemistry ,Chromatography, Affinity ,chemistry.chemical_compound ,Mice ,In vivo ,Ribose ,Methods ,Animals ,Molecular Biology ,Cells, Cultured ,Chromatography, High Pressure Liquid ,Chemistry ,Cell Biology ,Metabolism ,NAD ,In vitro ,Rats ,Cell Transformation, Neoplastic ,Liver ,Electrophoresis, Polyacrylamide Gel ,Poly(ADP-ribose) Polymerases - Published
- 1995
39. Histone Deposition at the Replication Fork
- Author
-
Alain Verreault
- Subjects
Genetics ,Nucleosome assembly ,Control of chromosome duplication ,Histone H2A ,Histone code ,Origin recognition complex ,Eukaryotic DNA replication ,Cell Biology ,Biology ,Pre-replication complex ,Molecular Biology ,S phase ,Cell biology - Abstract
In this issue of Molecular Cell, Ye et al. provide a biological rationale for rapid histone deposition behind the replication fork. They show that defects in nucleosome assembly lead to DNA double-strand breaks and S phase arrest. Their results have important implications for the maintenance of genome integrity in proliferating cells.
- Published
- 2003
- Full Text
- View/download PDF
40. When enough is enough: Detrimental effects of excess histones
- Author
-
Valérie Villeneuve and Alain Verreault
- Subjects
DNA metabolism ,chemistry.chemical_compound ,Histone ,biology ,Biochemistry ,chemistry ,biology.protein ,DNA replication ,Cell Biology ,Molecular Biology ,Dna packaging ,DNA ,Developmental Biology - Published
- 2010
41. Erratum: Fungal Rtt109 histone acetyltransferase is an unexpected structural homolog of metazoan p300/CBP
- Author
-
Philip A. Cole, Ronen Marmorstein, Pierre Thibault, Walter Rocha, Yong Tang, Alain Verreault, Katrina Meeth, Marc A. Holbert, Hugo Wurtele, Marlene Gharib, and Eva Jiang
- Subjects
Genetics ,biology ,Structural Biology ,biology.protein ,Histone acetyltransferase ,Molecular Biology ,Molecular biology - Published
- 2008
42. Purification and partial amino acid sequence of a glutamyl-tRNA synthetase from Rhizobium meliloti
- Author
-
Alexander W. Bell, Manon Belair, Serge Laberge, Jacques Lapointe, Alain Verreault, and Lucien M. Bordeleau
- Subjects
Molecular Sequence Data ,Peptide ,medicine.disease_cause ,Biochemistry ,Chromatography, Affinity ,Amino Acyl-tRNA Synthetases ,chemistry.chemical_compound ,Biosynthesis ,Bacterial Proteins ,Species Specificity ,medicine ,Amino Acid Sequence ,Cyanogen Bromide ,Molecular Biology ,Escherichia coli ,Peptide sequence ,chemistry.chemical_classification ,biology ,Cell Biology ,biology.organism_classification ,Chromatography, Ion Exchange ,Amino acid ,Glutamate-tRNA Ligase ,Enzyme ,chemistry ,Transfer RNA ,Chromatography, Gel ,bacteria ,Rhizobium ,Cell Division ,Chromatography, Liquid - Abstract
A glutamyl-tRNA synthetase has been purified to homogeneity from Rhizobium meliloti, using reversed-phase chromatography as the last step. Amino acid sequencing of the amino-terminal region of the enzyme indicates that it contains a single polypeptide, whose molecular weight is about 54 000, as judged by SDS–gel electrophoresis. The primary structures of the amino-terminus region and of an internal peptide obtained by cleavage of the enzyme with CNBr have similarities of 58 and 48% with regions of the glutamyl-tRNA sythetase of Escherichia coli; these are thought to be involved in the binding of ATP and tRNA, respectively. The small amount of glutamyl-tRNA synthetase present in R. meliloti is consistent with the metabolic regulation of the biosynthesis of many aminoacyl-tRNA synthetases.Key words: glutamyl-tRNA synthetase, Rhizobium meliloti, purification, reverse-phase chromatography, amino acid sequence.
- Published
- 1989
43. Hétérochromatine : un silence bien bruyant
- Author
-
Alain Verreault
- Subjects
Physics ,General Medicine ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology - Abstract
> Les sequences d’ADN repetitives adjacentes aux centromeres sont assemblees sous une forme speciale de chromatine, l’heterochromatine pericentrique, qui est generalement consideree comme une region chromosomique inerte. Cette reputation lui vient en partie du fait qu’elle impose un silence transcriptionnel aux genes qui y sont relocalises en raison de translocations chromosomiques. L’etat de ces genes maintenus reprimes sous l’influence de l’heterochromatine est transmis de facon stable a travers la mitose et la meiose. En accord avec ce caractere inerte et hostile a la transcription, l’heterochromatine pericentrique demeure condensee et relativement inaccessible aux nucleases pendant tout le cycle cellulaire. Les proteines HP1 (heterochromatin protein 1) sont une des composantes essentielles de l’heterochromatine pericentrique. Ces proteines sont depourvues de domaine de liaison a l’ADN. Leur association avec l’heterochromatine depend de leur domaine amino-terminal (chromo) qui se fixe de facon tres stable (Kd = 2mM) a l’histone H3 methylee sur le groupement e-amine de la lysine 9 (Lys 9) [1]. La presence d’un domaine de dimerisation dans sa partie carboxy-terminale (shadow chromo) permet a HP1 de fixer simultanement deux molecules d’H3 methylees sur la Lys9 de maniere intraou inter-nucleosomique (Figure 1A) (‹). Leur structure dimerique confere donc aux proteines HP1 le potentiel de compacter la chromatine en joignant des nucleosomes qui ne sont pas necessairement adjacents le long de la fibre de chromatine. La capacite de ponter deux fibres de chromatine distinctes pourrait aussi etre a l’origine de la relocalisation de certains genes au sein de l’heterochromatine pericentrique dans les cellules ou ces genes sont sujets a une repression transcriptionnelle [2]. Cette propriete des proteines HP1 pourrait aussi servir a renforcer la cohesion pericentromerique pour resister aux forces de tension du fuseau mitotique et empecher la dissociation prematuree des chromatides sœurs durant la mitose [3]. Contre toute attente, deux etudes recentes revelent que les proteines HP1 sont en fait associees de facon tres dynamique avec l’heterochromatine pericentrique [4, 5]. Ces etudes font appel a une technique de biologie cellulaire qui permet de mesurer la mobilite de proteines de fusion GFP (green fluorescent protein) in vivo. Cette technique, appelee «retour de fluorescence apres photoblanchiment» (fluorescence NOUVELLE
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