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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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Mcl1 localizes to replication foci during S-phase.(A) Mcl1 changes its nuclear localization during S-phase. Cells expressing Mcl1-Venus from the endogenous locus were classified into 4 cell cycle stages according to their morphology. The percentage of cells showing punctate (closed bar), condensed (shaded bar), and dispersed (open bar) localization of Mcl1-Venus in various cell cycle stages are shown (left panel). Representative photographs of each localization pattern are shown (right panel). (B–D) Mcl1 and Swi7 colocalize with PCNA in the S-phase nucleus. Mcl1-GFP and ECFP-PCNA (B), Swi7-GFP and ECFP-PCNA (C), and Swi7-Venus and Mcl1-ECFP (D) are shown. Merged images are also shown.
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pone-0002221-g007: Mcl1 localizes to replication foci during S-phase.(A) Mcl1 changes its nuclear localization during S-phase. Cells expressing Mcl1-Venus from the endogenous locus were classified into 4 cell cycle stages according to their morphology. The percentage of cells showing punctate (closed bar), condensed (shaded bar), and dispersed (open bar) localization of Mcl1-Venus in various cell cycle stages are shown (left panel). Representative photographs of each localization pattern are shown (right panel). (B–D) Mcl1 and Swi7 colocalize with PCNA in the S-phase nucleus. Mcl1-GFP and ECFP-PCNA (B), Swi7-GFP and ECFP-PCNA (C), and Swi7-Venus and Mcl1-ECFP (D) are shown. Merged images are also shown.

Mentions: Mcl1 was previously shown to be a constitutive nuclear protein that associates with chromatin from G1 to S phase [28]. To further examine the nuclear localization of Mcl1, the endogenous mcl1+ gene was C-terminally tagged with the fluorescent marker Venus [38]. The mcl1+-venus cell did not show any phenotypes such as HU and MMS sensitivity (data not shown). Mcl1-Venus localized to the nucleus throughout the cell cycle, and a portion of cells showed a punctate (Figure 7A, top) or condensed pattern (Figure 7A, middle) of Mcl1-Venus signal. To determine which stage of the cell cycle shows such localization pattern, mcl1+-venus cells were stained with Hoechst 33432 to visualize nucleus and septum, and roughly classified into four cell cycle stages (Figure 7A). A diffuse Mcl1-Venus signal was observed in the nucleus of most cells with a single nucleus (G2-phase). Condensed localization of Mcl1-Venus peaked in cells with a septum (G1/S-phase) and decreased in separating cells (S/G2-phase). The punctate localization of Mcl1-Venus increased from G1/S-phase and accounted for about half of S/G2 cells. These results indicate that Mcl1 changes its nuclear localization pattern during DNA replication.


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)

Mcl1 localizes to replication foci during S-phase.(A) Mcl1 changes its nuclear localization during S-phase. Cells expressing Mcl1-Venus from the endogenous locus were classified into 4 cell cycle stages according to their morphology. The percentage of cells showing punctate (closed bar), condensed (shaded bar), and dispersed (open bar) localization of Mcl1-Venus in various cell cycle stages are shown (left panel). Representative photographs of each localization pattern are shown (right panel). (B–D) Mcl1 and Swi7 colocalize with PCNA in the S-phase nucleus. Mcl1-GFP and ECFP-PCNA (B), Swi7-GFP and ECFP-PCNA (C), and Swi7-Venus and Mcl1-ECFP (D) are shown. Merged images are also shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2376062&req=5

pone-0002221-g007: Mcl1 localizes to replication foci during S-phase.(A) Mcl1 changes its nuclear localization during S-phase. Cells expressing Mcl1-Venus from the endogenous locus were classified into 4 cell cycle stages according to their morphology. The percentage of cells showing punctate (closed bar), condensed (shaded bar), and dispersed (open bar) localization of Mcl1-Venus in various cell cycle stages are shown (left panel). Representative photographs of each localization pattern are shown (right panel). (B–D) Mcl1 and Swi7 colocalize with PCNA in the S-phase nucleus. Mcl1-GFP and ECFP-PCNA (B), Swi7-GFP and ECFP-PCNA (C), and Swi7-Venus and Mcl1-ECFP (D) are shown. Merged images are also shown.
Mentions: Mcl1 was previously shown to be a constitutive nuclear protein that associates with chromatin from G1 to S phase [28]. To further examine the nuclear localization of Mcl1, the endogenous mcl1+ gene was C-terminally tagged with the fluorescent marker Venus [38]. The mcl1+-venus cell did not show any phenotypes such as HU and MMS sensitivity (data not shown). Mcl1-Venus localized to the nucleus throughout the cell cycle, and a portion of cells showed a punctate (Figure 7A, top) or condensed pattern (Figure 7A, middle) of Mcl1-Venus signal. To determine which stage of the cell cycle shows such localization pattern, mcl1+-venus cells were stained with Hoechst 33432 to visualize nucleus and septum, and roughly classified into four cell cycle stages (Figure 7A). A diffuse Mcl1-Venus signal was observed in the nucleus of most cells with a single nucleus (G2-phase). Condensed localization of Mcl1-Venus peaked in cells with a septum (G1/S-phase) and decreased in separating cells (S/G2-phase). The punctate localization of Mcl1-Venus increased from G1/S-phase and accounted for about half of S/G2 cells. These results indicate that Mcl1 changes its nuclear localization pattern during DNA replication.

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