Biology subjects

Ikui, A. E.

Publications and source records attributed to Ikui, A. E..

4 recordsLinked to original sources

bioRxiv2021

Cdc6 is sequentially regulated by PP2A-Cdc55, Cdc14 and Sic1 for origin licensing in S. cerevisiae

Cdc6, a subunit of the pre-replicative complex, contains multiple regulatory Cdk1 consensus sites, SP or TP motifs. In S. cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S-phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the Cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, the Cdk1 inhibitor, Sic1, releases Clb2{middle dot}Cdk1{middle dot}Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing is promoted by the attenuation of distinct CDK-dependent Cdc6 inhibitory mechanisms.

cell biology↗

bioRxiv2020

PP2A-Cdc55 is responsible for mitotic arrest in DNA re-replicating cells in S. cerevisiae

The cell cycle is an ordered process in which cells replicate their DNA in S-phase and divide them into two identical daughter cells in mitosis. DNA replication takes place only once per cell cycle to preserve genome integrity, which is tightly regulated by Cyclin Dependent Kinase (CDK). Formation of the pre-replicative complex, a platform for origin licensing, is inhibited through CDK-dependent phosphorylation. Failure of this control leads to re-licensing, re-replication and DNA damage. Eukaryotic cells have evolved surveillance mechanisms to maintain genome integrity, termed cell cycle checkpoints. It has been shown that the DNA damage checkpoint is activated upon the induction of DNA re-replication and arrests cell cycle in mitosis in S. cerevisiae. In this study, we show that PP2A-Cdc55 is responsible for the metaphase arrest induced by DNA re-replication, leading to dephosphorylation of APC component, Exclusion of Cdc55 from the nucleus bypassed the mitotic arrest and resulted in enhanced cell lethality in re-replicating cells. The metaphase arrest in re-replication cells was retained in the absence of Mad2, a key component of the spindle assembly checkpoint. Moreover, re-replicating cells showed the same rate of DNA damage induction in the presence or absence of Cdc55. These results indicate that PP2A-Cdc55 maintains metaphase arrest upon DNA re-replication and DNA damage through APC inhibition.

cell biology↗

bioRxiv2020

Conservation and divergence of DNA replication control in Chlamydomonas reinhardtii

We recently isolated temperature-sensitive cell cycle mutants in Chlamydomonas reinhardtii for which the causative mutations were located in genes annotated for potential involvement in DNA replication. Chlamydomonas has a very long G1 period during which cells grow up to ~10-fold without division, followed by rapid cycles of DNA replication and mitosis ( multiple fission). All of the candidate DNA replication mutants tested were defective in completion of the first round of DNA replication, and also failed to produce mitotic spindles. For a subset of the mutants, we rescued temperature-sensitive lethality with tagged transgenes and used the resulting strains to analyze abundance and localization control of the tagged protein. All of the DNA replication proteins tested were essentially undetectable until late G1, accumulated during the period of multiple fission and then were degraded as cells completed their terminal divisions. MCM4 and MCM6 were localized to the nucleus during the division cycle except for transient cytoplasmic localization during mitosis. CDC45 showed strict protein location to the nucleus and co-localized to spindles during mitosis. In contrast, CDC6 was detected in the nucleus only transiently during early divisions within the overall multiple fission cycle. Cdc6 protein levels were very low, but increased upon treatment with MG132, a proteasome inhibitor. We also tested if these DNA replication proteins are regulated by cyclin dependent kinase (CDK). There are two main CDKs in Chlamydomonas, CDKA1 and CDKB1. We found that CDC6 protein level was severely reduced in a cdka1 mutant, but not in a cdkb1 mutant. MG132 did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 upregulates CDC6 at the transcription level. Since MCM4, MCM6 and CDC6 were all essentially undetectable during the long G1 before DNA replication cycles began, we speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. These results provide a microbial framework for approaching replication control in the plant kingdom.

plant biology↗

bioRxiv2019

PP2ACdc55 dephosphorylates Pds1 to inhibit spindle elongation

DNA replication stress stalls replication forks leading to chromosome breakage and Intra-S checkpoint activation. In S. cerevisiae, this checkpoint arrests the cell cycle by stabilizing securin (Pds1) and inhibiting the cyclin dependent kinase (CDK) through multiple pathways. Pds1 inhibits separase (Esp1) which cleaves the cohesin subunit Scc1 and also functions in spindle elongation. However, the role of Pds1-Esp1 in spindle elongation during replication stress response is unknown. Here, we show that Pds1 phosphorylation plays a positive role in spindle elongation through the Pds1-Esp1 interaction in unperturbed and replication stress conditions. PP2ACdc55 directly dephosphorylates Pds1 both in vivo and in vitro. Pds1 hyperphosphorylation in a cdc55{Delta} mutant enhanced the Pds1-Esp1 interaction, which accelerated spindle elongation. This PP2ACdc55-dependent Pds1 dephosphorylation plays a role during replication stress and acts independently of the known Mec1, Swe1 or Spindle Assembly Checkpoint (SAC) checkpoint pathways. We propose a model where PP2ACdc55 dephosphorylates Pds1 to disrupt the Pds1-Esp1 interaction that inhibits spindle elongation during replication stress.

cell biology↗