scholarly journals Mutational Analysis of the Sir3 BAH Domain Reveals Multiple Points of Interaction with Nucleosomes

2009 ◽  
Vol 29 (10) ◽  
pp. 2532-2545 ◽  
Author(s):  
Vinaya Sampath ◽  
Peihua Yuan ◽  
Isabel X. Wang ◽  
Evelyn Prugar ◽  
Fred van Leeuwen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutational Analysis ◽  
Histone H3 ◽  
Transcriptional Silencing ◽  
In Vitro Experiments ◽  
Multiple Points ◽  
Alanine Residue ◽  
Bah Domain ◽  
Multiple Domains

ABSTRACT Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.

scholarly journals Structure and Function of the Saccharomyces cerevisiae Sir3 BAH Domain

2006 ◽  
Vol 26 (8) ◽  
pp. 3256-3265 ◽  
Author(s):  
Jessica J. Connelly ◽  
Peihua Yuan ◽  
Hao-Chi Hsu ◽  
Zhizhong Li ◽  
Rui-Ming Xu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein A ◽  
Transcriptional Silencing ◽  
Structure And Function ◽  
Full Length ◽  
Left Handed ◽  
N Terminus ◽  
Bah Domain ◽  
And Function

ABSTRACT 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.

Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein

1989 ◽  
Vol 9 (11) ◽  
pp. 5034-5044
Author(s):  
J L Celenza ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Mutational Analysis ◽  
Gene Dosage ◽  
Regulatory System ◽  
Kinase Domain ◽  
Regulatory Function ◽  
Protein Kinase Activity ◽  
Glucose Limitation

The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

Yeast intragenic transcriptional control: activation and repression sites within the coding region of the Saccharomyces cerevisiae LPD1 gene

1994 ◽  
Vol 14 (1) ◽  
pp. 214-225
Author(s):  
D A Sinclair ◽  
G D Kornfeld ◽  
I W Dawes
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Rna Polymerase Ii ◽  
Mutational Analysis ◽  
Lacz Fusion ◽  
Dependent Manner ◽  
Coding Region ◽  
Coding Sequence

Though widely recognized in higher eukaryotes, the regulation of Saccharomyces cerevisiae genes transcribed by RNA polymerase II by proteins that bind within the coding sequence remains largely speculative. We have shown for the LPD1 gene, encoding lipoamide dehydrogenase, that the coding sequence between +13 and +469 activated gene expression of an LPD1::lacZ fusion by up to sixfold in the presence of the upstream promoter. This downstream region, inserted upstream of a promoterless CYC1::lacZ fusion, activated gene expression in a carbon source-dependent manner by a factor of 15 to 111, independent of orientation. Deletion and mutational analysis identified two downstream activation sites (DAS1 and DAS2) and two downstream repressor sites (DRS1 and DRS2) that influence the rate of LPD1 transcription rather than mRNA degradation or translation. Activation from the DAS1 region (positions +137 to +191), encompassing a CDEI-like element, is twofold under derepressive conditions. Activation from DAS2 (+291 to +296), a CRE-like motif, is 12-fold for both repressed and derepressed states. DRS1, a pair of adjacent and opposing ABF1 sites (+288 to +313), is responsible for a 1.3- to 2-fold repression of transcription, depending on the carbon source. DRS1 requires the concerted action of DRS2 (a RAP1 motif at position +406) for repression of transcription only when the gene is induced. Gel mobility shift analysis and in vitro footprinting have shown that proteins bind in vitro to these downstream elements.

scholarly journals Nicotinamide Clearance by Pnc1 Directly Regulates Sir2-Mediated Silencing and Longevity

2004 ◽  
Vol 24 (3) ◽  
pp. 1301-1312 ◽  
Author(s):  
Christopher M. Gallo ◽  
Daniel L. Smith ◽  
Jeffrey S. Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nicotinic Acid ◽  
Life Span ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Transcriptional Silencing ◽  
Inhibitory Effect ◽  
Salvage Pathway

ABSTRACT The Saccharomyces cerevisiae Sir2 protein is an NAD+-dependent histone deacetylase (HDAC) that functions in transcriptional silencing and longevity. The NAD+ salvage pathway protein, Npt1, regulates Sir2-mediated processes by maintaining a sufficiently high intracellular NAD+ concentration. However, another NAD+ salvage pathway component, Pnc1, modulates silencing independently of the NAD+ concentration. Nicotinamide (NAM) is a by-product of the Sir2 deacetylase reaction and is a natural Sir2 inhibitor. Pnc1 is a nicotinamidase that converts NAM to nicotinic acid. Here we show that recombinant Pnc1 stimulates Sir2 HDAC activity in vitro by preventing the accumulation of NAM produced by Sir2. In vivo, telomeric, rDNA, and HM silencing are differentially sensitive to inhibition by NAM. Furthermore, PNC1 overexpression suppresses the inhibitory effect of exogenously added NAM on silencing, life span, and Hst1-mediated transcriptional repression. Finally, we show that stress suppresses the inhibitory effect of NAM through the induction of PNC1 expression. Pnc1, therefore, positively regulates Sir2-mediated silencing and longevity by preventing the accumulation of intracellular NAM during times of stress.

scholarly journals The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase.

1994 ◽  
Vol 14 (12) ◽  
pp. 8282-8291 ◽  
Author(s):  
B T Roberts ◽  
K A Farr ◽  
M A Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Normal Cell ◽  
Kinase Activity ◽  
Cell Multiplication ◽  
In Vitro Experiments ◽  
Microtubule Inhibitor ◽  
Cycle Progression ◽  
Novel Protein

Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.

scholarly journals The histone H3-H4 tetramer is a copper reductase enzyme

Science ◽  
2020 ◽  
Vol 369 (6499) ◽  
pp. 59-64 ◽  
Author(s):  
Narsis Attar ◽  
Oscar A. Campos ◽  
Maria Vogelauer ◽  
Chen Cheng ◽  
Yong Xue ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Histone H3 ◽  
Copper Binding ◽  
Active Site Residues ◽  
Chromatin Compaction ◽  
Putative Active Site ◽  
Dimerization Interface ◽  
Cellular Copper

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3′ dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae. The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.

scholarly journals Histone H3 K4 Demethylation during Activation and Attenuation of GAL1 Transcription in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (22) ◽  
pp. 7856-7864 ◽  
Author(s):  
Kristin Ingvarsdottir ◽  
Chris Edwards ◽  
Min Gyu Lee ◽  
Jung Shin Lee ◽  
David C. Schultz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Gene Activation ◽  
Histone H3 ◽  
Histone Lysine ◽  
Insight Into ◽  
Demethylation Activity

ABSTRACT In mammalian cells, histone lysine demethylation is carried out by two classes of enzymes, the LSD1/BHC110 class and the jumonji class. The enzymes of the jumonji class in the yeast Saccharomyces cerevisiae have recently also been shown to have lysine demethylation activity. Here we report that the protein encoded by YJR119c (termed KDM5), coding for one of five predicted jumonji domain proteins in yeast, specifically demethylates trimethylated histone H3 lysine 4 (H3K4me3), H3K4me2, and H3K4me1 in vitro. We found that loss of KDM5 increased mono-, di-, and trimethylation of lysine 4 during activation of the GAL1 gene. Interestingly, cells deleted of KDM5 also displayed a delayed reduction of K4me3 upon reestablishment of GAL1 repression. These results indicate that K4 demethylation has two roles at GAL1, first to establish appropriate levels of K4 methylation during gene activation and second to remove K4 trimethylation during the attenuation phase of transcription. Thus, analysis of lysine demethylation in yeast provides new insight into the physiological roles of jumonji demethylase enzymes.

scholarly journals Chaperone Control of the Activity and Specificity of the Histone H3 Acetyltransferase Rtt109

2008 ◽  
Vol 28 (13) ◽  
pp. 4342-4353 ◽  
Author(s):  
Jeffrey Fillingham ◽  
Judith Recht ◽  
Andrea C. Silva ◽  
Bernhard Suter ◽  
Andrew Emili ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Acetyltransferase ◽  
Histone H3 ◽  
Histone Chaperone ◽  
Genetic Interactions ◽  
Whole Genome ◽  
Genome Screen ◽  
Associated Lesions

ABSTRACT Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109Δ suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers diacetylated on both H4-K5/12 and H3-K9/56.

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