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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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Multi-copy suppression of mcl1-101 phenotypes by sir2+ and clr3+ HDAC genes.Wild-type and mcl1-101 mutant harboring the ura4+ insertion at the cnt1 were transformed with multi-copy plasmid carrying sir2+, clr3+, or both, respectively. Tenfold serial dilutions were spotted onto EMM-Leu and EMM-Leu containing 5-FOA (A) or EMM-Leu containing indicated concentration of TBZ (B). In panel A, sir2+ and, to a lesser extent, clr3+ partially suppressed 5-FOA sensitivity of the mcl1-101 mutant. In panel B, clr3+ partially suppressed TBZ sensitivity of the mcl1-101 mutant, and sir2+ also relieved the sensitivity only in the presence of clr3+.
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pone-0002221-g006: Multi-copy suppression of mcl1-101 phenotypes by sir2+ and clr3+ HDAC genes.Wild-type and mcl1-101 mutant harboring the ura4+ insertion at the cnt1 were transformed with multi-copy plasmid carrying sir2+, clr3+, or both, respectively. Tenfold serial dilutions were spotted onto EMM-Leu and EMM-Leu containing 5-FOA (A) or EMM-Leu containing indicated concentration of TBZ (B). In panel A, sir2+ and, to a lesser extent, clr3+ partially suppressed 5-FOA sensitivity of the mcl1-101 mutant. In panel B, clr3+ partially suppressed TBZ sensitivity of the mcl1-101 mutant, and sir2+ also relieved the sensitivity only in the presence of clr3+.

Mentions: Increased acetylation of histone H4 in the mcl1 mutant raised the possibility that Mcl1 might regulate HDAC(s) to maintain the hypoacetylated state of the kinetochore domain. The S. pombe genome encodes six HDACs that belong to three distinct classes. Among these HDACs, Clr3, Clr6 and Hst4 are required for transcriptional gene silencing at the central core region [35], [36]. In addition, the HDAC Sir2 preferentially localizes to the central core region [37]. To examine whether these HDACs are functionally related to Mcl1, multi-copy plasmids carrying each of the HDAC genes were introduced into cnt1:ura4+ mcl1-101 cells, and sensitivity to 5-FOA and TBZ, were examined. As shown in Figure 6A, alleviation of silencing in the mcl1 mutant was partially suppressed by multi-copy plasmids of sir2+ gene and, to a lesser extent, of the clr3+ gene. Furthermore, TBZ sensitivity was partially suppressed by the clr3+ plasmid and simultaneous introduction of sir2+ and clr3+ genes further relieved the sensitivity (Figure 6B). These results suggest that Mcl1 functionally interacts with Sir2 and Clr3 HDACs in maintaining centromere integrity.


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)

Multi-copy suppression of mcl1-101 phenotypes by sir2+ and clr3+ HDAC genes.Wild-type and mcl1-101 mutant harboring the ura4+ insertion at the cnt1 were transformed with multi-copy plasmid carrying sir2+, clr3+, or both, respectively. Tenfold serial dilutions were spotted onto EMM-Leu and EMM-Leu containing 5-FOA (A) or EMM-Leu containing indicated concentration of TBZ (B). In panel A, sir2+ and, to a lesser extent, clr3+ partially suppressed 5-FOA sensitivity of the mcl1-101 mutant. In panel B, clr3+ partially suppressed TBZ sensitivity of the mcl1-101 mutant, and sir2+ also relieved the sensitivity only in the presence of clr3+.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002221-g006: Multi-copy suppression of mcl1-101 phenotypes by sir2+ and clr3+ HDAC genes.Wild-type and mcl1-101 mutant harboring the ura4+ insertion at the cnt1 were transformed with multi-copy plasmid carrying sir2+, clr3+, or both, respectively. Tenfold serial dilutions were spotted onto EMM-Leu and EMM-Leu containing 5-FOA (A) or EMM-Leu containing indicated concentration of TBZ (B). In panel A, sir2+ and, to a lesser extent, clr3+ partially suppressed 5-FOA sensitivity of the mcl1-101 mutant. In panel B, clr3+ partially suppressed TBZ sensitivity of the mcl1-101 mutant, and sir2+ also relieved the sensitivity only in the presence of clr3+.
Mentions: Increased acetylation of histone H4 in the mcl1 mutant raised the possibility that Mcl1 might regulate HDAC(s) to maintain the hypoacetylated state of the kinetochore domain. The S. pombe genome encodes six HDACs that belong to three distinct classes. Among these HDACs, Clr3, Clr6 and Hst4 are required for transcriptional gene silencing at the central core region [35], [36]. In addition, the HDAC Sir2 preferentially localizes to the central core region [37]. To examine whether these HDACs are functionally related to Mcl1, multi-copy plasmids carrying each of the HDAC genes were introduced into cnt1:ura4+ mcl1-101 cells, and sensitivity to 5-FOA and TBZ, were examined. As shown in Figure 6A, alleviation of silencing in the mcl1 mutant was partially suppressed by multi-copy plasmids of sir2+ gene and, to a lesser extent, of the clr3+ gene. Furthermore, TBZ sensitivity was partially suppressed by the clr3+ plasmid and simultaneous introduction of sir2+ and clr3+ genes further relieved the sensitivity (Figure 6B). These results suggest that Mcl1 functionally interacts with Sir2 and Clr3 HDACs in maintaining centromere integrity.

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