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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

Analysis of the topoisomer distribution in different yeast plasmids in WT and sir2Δ cells. WT or sir2Δ cells, transformed with the specified plasmid (indicated in each square) were grown to exponential phase and circular DNA purified. 2D electrophoresis was then performed with two different concentrations of cloroquine: 10 and 30 µg/ml in the first and second dimension, respectively. After electrophoresis, DNA samples were transferred to a nitrocellulose filter and hybridized with the probes specified. The topoisomers are distributed along an arc-shaped trajectory. Negatively and positively supercoiled topoisomers are found in the left and right parts of the arc, respectively. Each square marked by a capital letter (A–E) shows a comparison between WT and sir2Δ strains for the specified plasmid. (B and E) show the same plasmid in WT and sir2Δ strains in the absence (A) or presence (E) of uracil in the growth medium. The ind values reported at the bottom of each topoisomer distribution indicate the increase of negative distribution evaluated by densitometric analysis.
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fig1: Analysis of the topoisomer distribution in different yeast plasmids in WT and sir2Δ cells. WT or sir2Δ cells, transformed with the specified plasmid (indicated in each square) were grown to exponential phase and circular DNA purified. 2D electrophoresis was then performed with two different concentrations of cloroquine: 10 and 30 µg/ml in the first and second dimension, respectively. After electrophoresis, DNA samples were transferred to a nitrocellulose filter and hybridized with the probes specified. The topoisomers are distributed along an arc-shaped trajectory. Negatively and positively supercoiled topoisomers are found in the left and right parts of the arc, respectively. Each square marked by a capital letter (A–E) shows a comparison between WT and sir2Δ strains for the specified plasmid. (B and E) show the same plasmid in WT and sir2Δ strains in the absence (A) or presence (E) of uracil in the growth medium. The ind values reported at the bottom of each topoisomer distribution indicate the increase of negative distribution evaluated by densitometric analysis.

Mentions: When ypGM1 was analyzed by 2D electrophoresis, we observed (Figure 1A) that the distribution of topoisomers from the sir2Δ strain was more negatively supercoiled compared to the one from the WT strain (note the leftward shift of the arc in sir2Δ compared to WT). When the topoisomer distribution was analyzed by densitometric scanning, we observed that in the sir2Δ strain, the increase of negative distribution (ind) was 1.8 times greater than in WT (each distribution is compared with the WT, assigned the standard value of 1); this value was calculated as described in the Materials and Methods section. In order to determine whether this effect was due to the presence of the NTS sequence in the plasmid, we extended our analysis to the vector alone (pRS316 without a specific insertion). In this case also, the distribution of DNA topoisomers from sir2Δ cells was more biased towards negative supercoiling than in WT cells, as shown in Figure 1B. As both ypGM1 and pRS316 contain a CEN6 and an ARSH4 replication origin, it is perhaps not surprising that the ind values in particular are very similar. In order to assess the influence of the centromere and replication origin sequences, we used a non-centromeric plasmid containing the 2 µ replication origin, pADH426 (35). In this case we also observed (Figure 1C) results similar to those reported for the plasmids just described with an ind value of 1.5. A more negatively supercoiled distribution of topoisomers in sir2Δ versus WT was also observed both with the yCP50 plasmid (36) carrying a different replication origin (ARS1) and a different centromeric sequence (CEN4 instead of CEN6) (Figure 1D), and with the p414GAL (34), Figure 5 (compare WT and sir2Δ samples) differing in the selectable marker (TRP1 instead of URA3 present in all the previous plasmids). Further evaluation of the selectable marker influence on plasmid topology has been performed on a LEU2 based plasmid (p415Gal) and a coherent result was obtained (data not shown). In all the plasmids so far studied, the alteration of DNA topology depends on the SIR2 gene but is independent of the presence of specific elements, such as ribosomal sequences, selectable markers, centromeres or replication origins. The increase in negative superhelical distribution for different plasmids ranged from 1.9- to 1.3-fold relative to WT.


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)

Analysis of the topoisomer distribution in different yeast plasmids in WT and sir2Δ cells. WT or sir2Δ cells, transformed with the specified plasmid (indicated in each square) were grown to exponential phase and circular DNA purified. 2D electrophoresis was then performed with two different concentrations of cloroquine: 10 and 30 µg/ml in the first and second dimension, respectively. After electrophoresis, DNA samples were transferred to a nitrocellulose filter and hybridized with the probes specified. The topoisomers are distributed along an arc-shaped trajectory. Negatively and positively supercoiled topoisomers are found in the left and right parts of the arc, respectively. Each square marked by a capital letter (A–E) shows a comparison between WT and sir2Δ strains for the specified plasmid. (B and E) show the same plasmid in WT and sir2Δ strains in the absence (A) or presence (E) of uracil in the growth medium. The ind values reported at the bottom of each topoisomer distribution indicate the increase of negative distribution evaluated by densitometric analysis.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Analysis of the topoisomer distribution in different yeast plasmids in WT and sir2Δ cells. WT or sir2Δ cells, transformed with the specified plasmid (indicated in each square) were grown to exponential phase and circular DNA purified. 2D electrophoresis was then performed with two different concentrations of cloroquine: 10 and 30 µg/ml in the first and second dimension, respectively. After electrophoresis, DNA samples were transferred to a nitrocellulose filter and hybridized with the probes specified. The topoisomers are distributed along an arc-shaped trajectory. Negatively and positively supercoiled topoisomers are found in the left and right parts of the arc, respectively. Each square marked by a capital letter (A–E) shows a comparison between WT and sir2Δ strains for the specified plasmid. (B and E) show the same plasmid in WT and sir2Δ strains in the absence (A) or presence (E) of uracil in the growth medium. The ind values reported at the bottom of each topoisomer distribution indicate the increase of negative distribution evaluated by densitometric analysis.
Mentions: When ypGM1 was analyzed by 2D electrophoresis, we observed (Figure 1A) that the distribution of topoisomers from the sir2Δ strain was more negatively supercoiled compared to the one from the WT strain (note the leftward shift of the arc in sir2Δ compared to WT). When the topoisomer distribution was analyzed by densitometric scanning, we observed that in the sir2Δ strain, the increase of negative distribution (ind) was 1.8 times greater than in WT (each distribution is compared with the WT, assigned the standard value of 1); this value was calculated as described in the Materials and Methods section. In order to determine whether this effect was due to the presence of the NTS sequence in the plasmid, we extended our analysis to the vector alone (pRS316 without a specific insertion). In this case also, the distribution of DNA topoisomers from sir2Δ cells was more biased towards negative supercoiling than in WT cells, as shown in Figure 1B. As both ypGM1 and pRS316 contain a CEN6 and an ARSH4 replication origin, it is perhaps not surprising that the ind values in particular are very similar. In order to assess the influence of the centromere and replication origin sequences, we used a non-centromeric plasmid containing the 2 µ replication origin, pADH426 (35). In this case we also observed (Figure 1C) results similar to those reported for the plasmids just described with an ind value of 1.5. A more negatively supercoiled distribution of topoisomers in sir2Δ versus WT was also observed both with the yCP50 plasmid (36) carrying a different replication origin (ARS1) and a different centromeric sequence (CEN4 instead of CEN6) (Figure 1D), and with the p414GAL (34), Figure 5 (compare WT and sir2Δ samples) differing in the selectable marker (TRP1 instead of URA3 present in all the previous plasmids). Further evaluation of the selectable marker influence on plasmid topology has been performed on a LEU2 based plasmid (p415Gal) and a coherent result was obtained (data not shown). In all the plasmids so far studied, the alteration of DNA topology depends on the SIR2 gene but is independent of the presence of specific elements, such as ribosomal sequences, selectable markers, centromeres or replication origins. The increase in negative superhelical distribution for different plasmids ranged from 1.9- to 1.3-fold relative to WT.

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