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SIR2 modifies histone H4-K16 acetylation and affects superhelicity in the ARS region of plasmid chromatin in Saccharomyces cerevisiae.

Chiani F, Di Felice F, Camilloni G - Nucleic Acids Res. (2006)

Bottom Line: Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells.This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids.A model proposing interference with the replication machinery is discussed.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Rome, Italy.

ABSTRACT
The mutation of the SIR2 gene in Saccharomyces cerevisiae has been associated with a series of different phenotypes including loss of transcriptional silencing, genome instability and replicative aging. Thus, the SIR2 gene product is an important constituent of the yeast cell. SIR2 orthologues and paralogues have been discovered in organisms ranging from bacteria to man, underscoring the pivotal role of this protein. Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells. This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids. Our results indicate that this new phenotype is due to the lack of Sir2p histone deacetylase activity in the sir2Delta strain, because only the H4-K16 residue of the histone octamer undergoes an alteration of its acetylation state. A model proposing interference with the replication machinery is discussed.

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Related in: MedlinePlus

Interpretive model to account for the topological effect of the sir2Δ mutation. In WT cells, a defined amount of the MCM complex is bound to DNA, producing the observed level of supercoiling (equilibrium level). When Sir2p is absent, plasmid DNA is overloaded with the MCM complex, leading to increased negative supercoiling. In a strain overexpressing Sir2p, the excess protein limits loading of the MCM complex, leading to positive supercoiling.
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fig7: Interpretive model to account for the topological effect of the sir2Δ mutation. In WT cells, a defined amount of the MCM complex is bound to DNA, producing the observed level of supercoiling (equilibrium level). When Sir2p is absent, plasmid DNA is overloaded with the MCM complex, leading to increased negative supercoiling. In a strain overexpressing Sir2p, the excess protein limits loading of the MCM complex, leading to positive supercoiling.

Mentions: Thus, the overall conclusion is that Sir2p limits MCM complex loading onto ARSs: in the sir2Δ mutant, overloading of the complex increases the unwinding of DNA, leading to negative supercoiling of plasmids (see model in Figure 7). Based on our demonstration that the only amino acid hyperacetylated in plasmid chromatin is H4-K16, we hypothesize that a specific histone deacetylase activity is involved in the reported phenotypes, and more specifically that H4-K16 hyperacetylation represents a signal for the recruitment of additional factor(s), such as the MCM complex. This is in agreement with previous data (18) showing that loss of Sir2p catalytic activity increases MCM loading on ARS sequences. We cannot eliminate the possibility that our results were due not only to the Sir2p histone-deacetylase activity on H4-K16, but also to its putative ability to deacetylate different proteins. In fact, Sir2p homologues in other species, including mammals, have been demonstrated to deacetylate non-histone proteins as well (60). A further interpretation of our data are that it might reflect direct deacetylation activity of Sir2p on the MCM complex, with the consequent alteration of its function in the assembly of the Pre-RC complex. In support of this hypothesis, it has been demonstrated that Mcm3p is acetylated in mammalian cells (61).


SIR2 modifies histone H4-K16 acetylation and affects superhelicity in the ARS region of plasmid chromatin in Saccharomyces cerevisiae.

Chiani F, Di Felice F, Camilloni G - Nucleic Acids Res. (2006)

Interpretive model to account for the topological effect of the sir2Δ mutation. In WT cells, a defined amount of the MCM complex is bound to DNA, producing the observed level of supercoiling (equilibrium level). When Sir2p is absent, plasmid DNA is overloaded with the MCM complex, leading to increased negative supercoiling. In a strain overexpressing Sir2p, the excess protein limits loading of the MCM complex, leading to positive supercoiling.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636471&req=5

fig7: Interpretive model to account for the topological effect of the sir2Δ mutation. In WT cells, a defined amount of the MCM complex is bound to DNA, producing the observed level of supercoiling (equilibrium level). When Sir2p is absent, plasmid DNA is overloaded with the MCM complex, leading to increased negative supercoiling. In a strain overexpressing Sir2p, the excess protein limits loading of the MCM complex, leading to positive supercoiling.
Mentions: Thus, the overall conclusion is that Sir2p limits MCM complex loading onto ARSs: in the sir2Δ mutant, overloading of the complex increases the unwinding of DNA, leading to negative supercoiling of plasmids (see model in Figure 7). Based on our demonstration that the only amino acid hyperacetylated in plasmid chromatin is H4-K16, we hypothesize that a specific histone deacetylase activity is involved in the reported phenotypes, and more specifically that H4-K16 hyperacetylation represents a signal for the recruitment of additional factor(s), such as the MCM complex. This is in agreement with previous data (18) showing that loss of Sir2p catalytic activity increases MCM loading on ARS sequences. We cannot eliminate the possibility that our results were due not only to the Sir2p histone-deacetylase activity on H4-K16, but also to its putative ability to deacetylate different proteins. In fact, Sir2p homologues in other species, including mammals, have been demonstrated to deacetylate non-histone proteins as well (60). A further interpretation of our data are that it might reflect direct deacetylation activity of Sir2p on the MCM complex, with the consequent alteration of its function in the assembly of the Pre-RC complex. In support of this hypothesis, it has been demonstrated that Mcm3p is acetylated in mammalian cells (61).

Bottom Line: Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells.This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids.A model proposing interference with the replication machinery is discussed.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Rome, Italy.

ABSTRACT
The mutation of the SIR2 gene in Saccharomyces cerevisiae has been associated with a series of different phenotypes including loss of transcriptional silencing, genome instability and replicative aging. Thus, the SIR2 gene product is an important constituent of the yeast cell. SIR2 orthologues and paralogues have been discovered in organisms ranging from bacteria to man, underscoring the pivotal role of this protein. Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells. This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids. Our results indicate that this new phenotype is due to the lack of Sir2p histone deacetylase activity in the sir2Delta strain, because only the H4-K16 residue of the histone octamer undergoes an alteration of its acetylation state. A model proposing interference with the replication machinery is discussed.

Show MeSH
Related in: MedlinePlus