Structure/Function Analysis of the Hif1 Histone Chaperone in Saccharomyces cerevisiae

Chromosome Segregation ◽

Genome Stability ◽

Function Analysis ◽

Molecular Genetic ◽

Histone Chaperone ◽

Chromatin Assembly ◽

Biological Processes ◽

Genetic Approach ◽

Yeast Saccharomyces Cerevisiae

Understanding the regulation of chromatin structure is a vital aspect of molecular biology regarding its influences on biological processes such as DNA replication, transcription (gene expression), DNA repair, chromosome segregation and recombination. In the budding yeast Saccharomyces cerevisiae, a histone chaperone called Hif1 has been found in the nuclei as having a functional role in chromatin assembly. Hif1 is a homolog of the human protein NASP that is involved in the maintenance of genome stability. Previously, Hif1 has been shown to physically interact with Hat1, Hat2 and H3/H4 to form the NuB4 complex directly involved in chromatin assembly. A molecular genetic approach was conducted to determine which domain of Hif1 is involved in the interaction with the HAT1 complex.

In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation

10.1128/mcb.11.10.5212-5221.1991 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5212-5221

Author(s):

B Jehn ◽

R Niedenthal ◽

J H Hegemann

Keyword(s):

Chromosome Segregation ◽

Artificial Chromosome ◽

Complete Information ◽

Saturation Mutagenesis ◽

Chromosome Loss ◽

Single Mutation ◽

Double Base

In the yeast Saccharomyces cerevisiae, the complete information needed in cis to specify a fully functional mitotic and meiotic centromere is contained within 120 bp arranged in the three conserved centromeric (CEN) DNA elements CDEI, -II, and -III. The 25-bp CDEIII is most important for faithful chromosome segregation. We have constructed single- and double-base substitutions in all highly conserved residues and one nonconserved residue of this element and analyzed the mitotic in vivo function of the mutated CEN DNAs, using an artificial chromosome. The effects of the mutations on chromosome segregation vary between wild-type-like activity (chromosome loss rate of 4.8 x 10(-4)) and a complete loss of CEN function. Data obtained by saturation mutagenesis of the palindromic core sequence suggest asymmetric involvement of the palindromic half-sites in mitotic CEN function. The poor CEN activity of certain single mutations could be improved by introducing an additional single mutation. These second-site suppressors can be found at conserved and nonconserved positions in CDEIII. Our suppression data are discussed in the context of natural CDEIII sequence variations found in the CEN sequences of different yeast chromosomes.

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

10.1128/mcb.15.10.5607 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5607-5617 ◽

Cited By ~ 66

Author(s):

H T Tran ◽

N P Degtyareva ◽

N N Koloteva ◽

A Sugino ◽

H Masumoto ◽

...

Keyword(s):

Genome Stability ◽

Genome Instability ◽

Temperature Sensitive ◽

Direct Repeats ◽

Dna Polymerase Delta ◽

Dna Double Strand Breaks ◽

Replication Slippage ◽

Random Sequences

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.119.3.583 ◽

1992 ◽

Vol 119 (3) ◽

pp. 583-593 ◽

Cited By ~ 182

Author(s):

R E Palmer ◽

D S Sullivan ◽

T Huffaker ◽

D Koshland

Keyword(s):

Chromosome Segregation ◽

Mother Cell ◽

Fold Increase ◽

Spindle Pole ◽

Spindle Orientation ◽

Temperature Sensitive ◽

Chromosome Movement ◽

Cytoskeletal Elements

In the yeast Saccharomyces cerevisiae, before the onset of anaphase, the spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud. This spindle orientation enables proper chromosome segregation to occur during anaphase, allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell. In this study, we synchronized a population of cells before the onset of anaphase such that > 90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations. Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in approximately 40-50% of the cells. When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100-200-fold increase in the frequency of binucleated cell bodies. Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation. These data are consistent with the model that proper spindle orientation is maintained by directly or indirectly tethering the astral microtubules to the actin cytoskeleton. After nuclear migration, but before anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle. The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the nonkinetochore spindle microtubules. These results suggest that astral microtubules are not only important for spindle orientation before anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement. Potential roles for this force in spindle assembly and orientation are discussed.

Heritable capture of heterochromatin dynamics in Saccharomyces cerevisiae

eLife ◽

10.7554/elife.05007 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 47

Author(s):

Anne E Dodson ◽

Jasper Rine

Keyword(s):

Chromosome Segregation ◽

Single Molecule ◽

Genome Stability ◽

Gene Repression ◽

Cell Divisions ◽

Rna Fish ◽

Eukaryotic Gene ◽

The Stability ◽

Lines Of Evidence

Heterochromatin exerts a heritable form of eukaryotic gene repression and contributes to chromosome segregation fidelity and genome stability. However, to date there has been no quantitative evaluation of the stability of heterochromatic gene repression. We designed a genetic strategy to capture transient losses of gene silencing in Saccharomyces as permanent, heritable changes in genotype and phenotype. This approach revealed rare transcription within heterochromatin that occurred in approximately 1/1000 cell divisions. In concordance with multiple lines of evidence suggesting these events were rare and transient, single-molecule RNA FISH showed that transcription was limited. The ability to monitor fluctuations in heterochromatic repression uncovered previously unappreciated roles for Sir1, a silencing establishment factor, in the maintenance and/or inheritance of silencing. In addition, we identified the sirtuin Hst3 and its histone target as contributors to the stability of the silenced state. These approaches revealed dynamics of a heterochromatin function that have been heretofore inaccessible.

Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids

10.1128/mcb.10.5.2176-2181.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2176-2181

Author(s):

R C Dickson ◽

G B Wells ◽

A Schmidt ◽

R L Lester

Keyword(s):

Membrane Lipids ◽

Molecular Genetic ◽

Suppressor Gene ◽

Molecular Genetic Analysis ◽

Yeast Cells ◽

Chain Base ◽

Long Chain Base ◽

Long Chain

Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.

The MCM16 gene of the yeast Saccharomyces cerevisiae is required for chromosome segregation

MGG Molecular & General Genetics ◽

10.1007/s004380050892 ◽

1998 ◽

Vol 260 (2-3) ◽

pp. 242-250 ◽

Cited By ~ 14

Author(s):

K. Sanyal ◽

S. K. Ghosh ◽

P. Sinha

Keyword(s):

Chromosome Segregation ◽

Yeast Saccharomyces Cerevisiae

In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation.

10.1128/mcb.11.10.5212 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5212-5221 ◽

Cited By ~ 26

Author(s):

B Jehn ◽

R Niedenthal ◽

J H Hegemann

Keyword(s):

Chromosome Segregation ◽

Artificial Chromosome ◽

Complete Information ◽

Saturation Mutagenesis ◽

Chromosome Loss ◽

Single Mutation ◽

Double Base

In the yeast Saccharomyces cerevisiae, the complete information needed in cis to specify a fully functional mitotic and meiotic centromere is contained within 120 bp arranged in the three conserved centromeric (CEN) DNA elements CDEI, -II, and -III. The 25-bp CDEIII is most important for faithful chromosome segregation. We have constructed single- and double-base substitutions in all highly conserved residues and one nonconserved residue of this element and analyzed the mitotic in vivo function of the mutated CEN DNAs, using an artificial chromosome. The effects of the mutations on chromosome segregation vary between wild-type-like activity (chromosome loss rate of 4.8 x 10(-4)) and a complete loss of CEN function. Data obtained by saturation mutagenesis of the palindromic core sequence suggest asymmetric involvement of the palindromic half-sites in mitotic CEN function. The poor CEN activity of certain single mutations could be improved by introducing an additional single mutation. These second-site suppressors can be found at conserved and nonconserved positions in CDEIII. Our suppression data are discussed in the context of natural CDEIII sequence variations found in the CEN sequences of different yeast chromosomes.

Assortment of Phosphatidylinositol 3-Kinase Complexes—Atg14p Directs Association of Complex I to the Pre-autophagosomal Structure in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e05-09-0841 ◽

2006 ◽

Vol 17 (4) ◽

pp. 1527-1539 ◽

Cited By ~ 151

Author(s):

Keisuke Obara ◽

Takayuki Sekito ◽

Yoshinori Ohsumi

Keyword(s):

Complex I ◽

Regulatory Subunit ◽

Biological Processes ◽

Phosphatidylinositol 3 Kinase ◽

Complex Ii ◽

Vacuolar Protein ◽

Vacuolar Membranes ◽

Phosphatidylinositol 3

In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.

The Epigenetic Pathways to Ribosomal DNA Silencing

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00005-16 ◽

2016 ◽

Vol 80 (3) ◽

pp. 545-563 ◽

Cited By ~ 25

Author(s):

Rakesh Srivastava ◽

Rashmi Srivastava ◽

Seong Hoon Ahn

Keyword(s):

Chromosome Segregation ◽

Ribosomal Dna ◽

Genome Stability ◽

Cellular Aging ◽

Content Type ◽

Eukaryotic Chromatin ◽

Normal Life Span ◽

Protein Components ◽

Dna Silencing

SUMMARYHeterochromatin is the transcriptionally repressed portion of eukaryotic chromatin that maintains a condensed appearance throughout the cell cycle. At sites of ribosomal DNA (rDNA) heterochromatin, epigenetic states contribute to gene silencing and genome stability, which are required for proper chromosome segregation and a normal life span. Here, we focus on recent advances in the epigenetic regulation of rDNA silencing inSaccharomyces cerevisiaeand in mammals, including regulation by several histone modifications and several protein components associated with the inner nuclear membrane within the nucleolus. Finally, we discuss the perturbations of rDNA epigenetic pathways in regulating cellular aging and in causing various types of diseases.