Methods designed for the identification and characterization ofin vitro andin vivo chromatin assembly mutants inSaccharomyces cerevisiae

Harkness, Troy A. A.; Arnason, Terra; Legrand, Charmaine; Lone, Ashley

doi:10.1251/bpo58

Cited by 5 publications

(7 citation statements)

References 31 publications

(35 reference statements)

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“…In our previous work, we identified two E3s required for mitotic chromatin assembly, the Apc5p subunit of the APC and Rsp5p (19,20). In this study, we asked whether the involvement of Apc5p and Rsp5p in chromatin assembly reflects an interrelationship between the two proteins.…”

Section: Discussionmentioning

confidence: 94%

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Novel Interaction between Apc5p and Rsp5p in an Intracellular Signaling Pathway in Saccharomyces cerevisiae

Arnason

Pisclevich²,

Dash³

et al. 2005

Eukaryot Cell

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The ubiquitin-targeting pathway is evolutionarily conserved and critical for many cellular functions. Recently, we discovered a role for two ubiquitin-protein ligases (E3s), Rsp5p and the Apc5p subunit of the anaphase-promoting complex (APC), in mitotic chromatin assembly in Saccharomyces cerevisiae. In the present study, we investigated whether Rsp5p and Apc5p interact in an intracellular pathway regulating chromatin remodeling. Our genetic studies strongly suggest that Rsp5p and Apc5p do interact and that Rsp5p acts upstream of Apc5p. Since E3 enzymes typically require the action of a ubiquitin-conjugating enzyme (E2), we screened E2 mutants for chromatin assembly defects, which resulted in the identification of Cdc34p and Ubc7p. Cdc34p is the E2 component of the SCF (Skp1p/Cdc53p/F-box protein). Therefore, we analyzed additional SCF mutants for chromatin assembly defects. Defective chromatin assembly extracts generated from strains harboring a mutation in the Cdc53p SCF subunit or a nondegradable SCF target, Sic1 ⌬phos , confirmed that the SCF was involved in mitotic chromatin assembly. Furthermore, we demonstrated that Ubc7p physically and genetically interacts with Rsp5p, suggesting that Ubc7p acts as an E2 for Rsp5p. However, rsp5 CA and ⌬ubc7 mutations had opposite genetic effects on apc5 CA and cdc34-2 phenotypes. Therefore, the antagonistic interplay between ⌬ubc7 and rsp5 CA , with respect to cdc34-2 and apc5 CA , indicates that the outcome of Rsp5p's interaction with Cdc34p and Apc5p may depend on the E2 interacting with Rsp5p.

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Section: Discussionmentioning

confidence: 94%

“…Benomyl was resuspended in 1% ethanol to a final concentration of 20 mg/ml and used at a final concentration of 12.5 g/ml. In vitro chromatin assembly assays were performed according to previously published protocols (19,20). Spot dilutions were conducted by first concentrating fresh overnight yeast cultures to 2 ϫ 10 7 cells/ml.…”

Section: Methodsmentioning

confidence: 99%

Novel Interaction between Apc5p and Rsp5p in an Intracellular Signaling Pathway in Saccharomyces cerevisiae

Arnason

Pisclevich²,

Dash³

et al. 2005

Eukaryot Cell

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“…Standard genetic techniques were performed as described (19). In vitro chromatin assembly assays were performed according to previously published protocols (20,21). Histone add-back experiments were conducted by including histones isolated from untreated or colcemid-treated HeLa cells, following acid extraction (Upstate), in chromatin assembly reactions according to the nonradioactive protocol (21).…”

Section: Methodsmentioning

confidence: 99%

“…Cells were grown in CA in vitro chromatin assembly defect. The transformants described above were assayed for in vitro chromatin assembly according to published protocols (20,21). The position of uniquely labeled, circular, relaxed pBlueScript plasmid (O, R) is indicated.…”

Section: Deletion Of Any Of the Caf-i Subunits Does Not Exacerbate Thmentioning

confidence: 99%

“…However, little is known regarding how CAFs ensure proper chromatin assembly or how they are regulated. Recently, we isolated the apc5 CA mutant, which compromised anaphase-promoting complex (APC) activity and impaired a novel mitotic chromatin assembly activity (20,21). Furthermore, we demonstrated that the APC is required in Saccharomyces cerevisiae for longevity (22).…”

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confidence: 99%

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Contribution of CAF-I to Anaphase-Promoting-Complex-Mediated Mitotic Chromatin Assembly in Saccharomyces cerevisiae

Harkness¹,

Arnason

Legrand³

et al. 2005

Eukaryot Cell

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The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5 CA mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5 CA phenotypes in a CAF-1-and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1⌬ cac2⌬ msi1⌬ (caf1⌬) cells expressing apc5 CA exhibit a phenotype more severe than that of apc5 CA or caf1⌬. The Ts ؊ phenotypes observed in apc5 CA and apc5 CA caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts ؊ defects. Synthetic genetic interactions were also observed in apc5 CA asf1⌬ cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5 CA Ts ؊ defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APCdependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5 CA phenotypes. We discuss the implications of our observations.

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Decondensation ofXenopussperm chromatin usingSaccharomyces cerevisiaewhole-cell extractsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Harkness

2006

Can. J. Physiol. Pharmacol.

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Biochemical studies using highly condensed Xenopus sperm chromatin and protein extracts prepared from multiple systems have lead to the identification of conserved proteins involved in chromosome decondensation. However, mutations to these proteins are unavailable as the systems used are not amenable to genetic studies. We took a genetic approach to isolating chromosome decondensation mutants by incubating Xenopus sperm chromatin with whole-cell extracts prepared from the Hartwell library of random temperature sensitive (ts) yeast cells. We show that decondensation of Xenopus sperm chromatin using wild type yeast extracts was rapid, ATP- and extract-dependent, and resistant to heat, N-ethylmaleimide, protease K, RNase A, and micrococcal nuclease. From 100 mutant extracts screened, we obtained one strain, referred to as rmc4, that was chromosome decondensation defective. The mutant was slow growing and exhibited germination defects. Low concentrations of rmc4 extract would eventually decondense sperm heads, and fractionation of the mutant extract produced a decondensation competent fraction, suggesting the presence of an overactive inhibitor in rmc4 cells. We performed a multicopy suppressor screen that identified PDE2, a gene encoding a protein that inhibits protein kinase A (PKA) activity. As PKA was previously shown in human cells to maintain condensed chromatin, our results suggest that PKA activity is elevated in rmc4 cells, causing a decondensation defect. Thus, our experiments reveal that yeast encodes an evolutionarily conserved chromosome decondensation activity that can be genetically manipulated.

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Methods designed for the identification and characterization ofin vitro andin vivo chromatin assembly mutants inSaccharomyces cerevisiae

Cited by 5 publications

References 31 publications

Novel Interaction between Apc5p and Rsp5p in an Intracellular Signaling Pathway in Saccharomyces cerevisiae

Novel Interaction between Apc5p and Rsp5p in an Intracellular Signaling Pathway in Saccharomyces cerevisiae

Contribution of CAF-I to Anaphase-Promoting-Complex-Mediated Mitotic Chromatin Assembly in Saccharomyces cerevisiae

Decondensation ofXenopussperm chromatin usingSaccharomyces cerevisiaewhole-cell extractsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

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