Analysis of Mcm2–7 chromatin binding during anaphase and in the transition to quiescence in fission yeast

Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2–7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.

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GINS Inactivation Phenotypes Reveal Two Pathways for Chromatin Association of Replicative α and ε DNA Polymerases in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e08-04-0429 ◽

2009 ◽

Vol 20 (4) ◽

pp. 1213-1222 ◽

Cited By ~ 16

Author(s):

Chen Chun Pai ◽

Ignacio García ◽

Shao Win Wang ◽

Sue Cotterill ◽

Stuart A. MacNeill ◽

...

Keyword(s):

Dna Replication ◽

Fission Yeast ◽

Dna Polymerase ◽

Dna Polymerases ◽

Chromatin Binding ◽

Dna Polymerase Α ◽

Replication Arrest ◽

Eukaryotic Dna ◽

Separate Pathway ◽

Hormone Binding Domain

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of β-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase ε, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase α is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase ε chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase α.

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