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A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

Natsume T, Tsutsui Y, Sutani T, Dunleavy EM, Pidoux AL, Iwasaki H, Shirahige K, Allshire RC, Yamao F - PLoS ONE (2008)

Bottom Line: The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains.Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha.These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Division of Mutagenesis, National Institute of Genetics, Mishima, Shizuoka, Japan.

ABSTRACT
Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

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The mcl1-101 and swi7-H4 mutants display alleviation of transcriptional gene silencing at both central core and outer repeat regions.(A) (upper panel) The schematic diagram of ura4+ and ade6+ genes inserted into imr1L and otr1R, respectively. Note that the ura4+ gene is inserted into heterochromatic domain, which is defined as outside of tRNA genes (vertical lines) [23]. (lower panel) Tenfold serial dilutions were spotted. (B) (upper panel) The schematic diagram of the ura4+ gene inserted into cnt1. (lower panel) Tenfold serial dilutions were spotted.
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pone-0002221-g003: The mcl1-101 and swi7-H4 mutants display alleviation of transcriptional gene silencing at both central core and outer repeat regions.(A) (upper panel) The schematic diagram of ura4+ and ade6+ genes inserted into imr1L and otr1R, respectively. Note that the ura4+ gene is inserted into heterochromatic domain, which is defined as outside of tRNA genes (vertical lines) [23]. (lower panel) Tenfold serial dilutions were spotted. (B) (upper panel) The schematic diagram of the ura4+ gene inserted into cnt1. (lower panel) Tenfold serial dilutions were spotted.

Mentions: To determine whether the mcl1-101 has defects in centromeric silencing like the other mcl1 mutant alleles, we introduced the mcl1-101 mutation into indicator strains. Strain FY1193 harbors the ura4+ and ade6+ genes inserted in the outer repeat imr and otr elements of cen1, respectively [19]. The mcl1-101 mutant harboring maker genes at the centromere was more sensitive to 5-FOA and was pink on YE low adenine plate compared to the wild-type strain which gave a red color (Figure 3A). The level of alleviation in mcl1-101 cells was comparable to that of swi6Δ cells at imr (FOA), but lower at otr (low ade). Previously we have revealed strong interaction between mcl1-101 and swi7-H4, a temperature sensitive allele of swi7+ [29]. Therefore we tested transcriptional gene silencing at heterochromatic regions in this mutant. As reported previously [30], swi7-H4 cells showed alleviation of transcriptional gene silencing at heterochromatic regions (Figure 3A). No dramatic reduction of the heterochromatic marks Swi6 or H3K9me2 on centromeric sequences was apparent in mcl1 mutants (T.N., E.D., A.P., Y.T., and R.A., unpublished data). Therefore, Mcl1 might function at this domain either independently or downstream of Swi6. Recently, Mamnun et al. isolated Mcl1 as an interacting partner of the F-box protein, Pof3, which is a substrate adapter of the SCF ubiquitin ligase and is required for genome integrity. They reported that only a slight change in Swi6-GFP localization could be detected in an mcl1Δ mutant [31], consistent with our observations. As the mcl1-101 mutation genetically interact with rad2Δ or dna2-C2 mutations [29], we examined transcriptional gene silencing in these mutants. However, these mutants did not show any defect in heterochromatin silencing (data not shown), suggesting that Mcl1 and Swi7 might have a specific role in centromeric chromatin structures.


A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

Natsume T, Tsutsui Y, Sutani T, Dunleavy EM, Pidoux AL, Iwasaki H, Shirahige K, Allshire RC, Yamao F - PLoS ONE (2008)

The mcl1-101 and swi7-H4 mutants display alleviation of transcriptional gene silencing at both central core and outer repeat regions.(A) (upper panel) The schematic diagram of ura4+ and ade6+ genes inserted into imr1L and otr1R, respectively. Note that the ura4+ gene is inserted into heterochromatic domain, which is defined as outside of tRNA genes (vertical lines) [23]. (lower panel) Tenfold serial dilutions were spotted. (B) (upper panel) The schematic diagram of the ura4+ gene inserted into cnt1. (lower panel) Tenfold serial dilutions were spotted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376062&req=5

pone-0002221-g003: The mcl1-101 and swi7-H4 mutants display alleviation of transcriptional gene silencing at both central core and outer repeat regions.(A) (upper panel) The schematic diagram of ura4+ and ade6+ genes inserted into imr1L and otr1R, respectively. Note that the ura4+ gene is inserted into heterochromatic domain, which is defined as outside of tRNA genes (vertical lines) [23]. (lower panel) Tenfold serial dilutions were spotted. (B) (upper panel) The schematic diagram of the ura4+ gene inserted into cnt1. (lower panel) Tenfold serial dilutions were spotted.
Mentions: To determine whether the mcl1-101 has defects in centromeric silencing like the other mcl1 mutant alleles, we introduced the mcl1-101 mutation into indicator strains. Strain FY1193 harbors the ura4+ and ade6+ genes inserted in the outer repeat imr and otr elements of cen1, respectively [19]. The mcl1-101 mutant harboring maker genes at the centromere was more sensitive to 5-FOA and was pink on YE low adenine plate compared to the wild-type strain which gave a red color (Figure 3A). The level of alleviation in mcl1-101 cells was comparable to that of swi6Δ cells at imr (FOA), but lower at otr (low ade). Previously we have revealed strong interaction between mcl1-101 and swi7-H4, a temperature sensitive allele of swi7+ [29]. Therefore we tested transcriptional gene silencing at heterochromatic regions in this mutant. As reported previously [30], swi7-H4 cells showed alleviation of transcriptional gene silencing at heterochromatic regions (Figure 3A). No dramatic reduction of the heterochromatic marks Swi6 or H3K9me2 on centromeric sequences was apparent in mcl1 mutants (T.N., E.D., A.P., Y.T., and R.A., unpublished data). Therefore, Mcl1 might function at this domain either independently or downstream of Swi6. Recently, Mamnun et al. isolated Mcl1 as an interacting partner of the F-box protein, Pof3, which is a substrate adapter of the SCF ubiquitin ligase and is required for genome integrity. They reported that only a slight change in Swi6-GFP localization could be detected in an mcl1Δ mutant [31], consistent with our observations. As the mcl1-101 mutation genetically interact with rad2Δ or dna2-C2 mutations [29], we examined transcriptional gene silencing in these mutants. However, these mutants did not show any defect in heterochromatin silencing (data not shown), suggesting that Mcl1 and Swi7 might have a specific role in centromeric chromatin structures.

Bottom Line: The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains.Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha.These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Division of Mutagenesis, National Institute of Genetics, Mishima, Shizuoka, Japan.

ABSTRACT
Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

Show MeSH
Related in: MedlinePlus