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Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing.

ABSTRACT: In a search for Polo-like kinase 1 (Plk1)-interacting proteins using a yeast two-hybrid system, we have identified histone acetyltransferase binding to the origin recognition complex 1 (Hbo1) as a potential Plk1 target. Here, we show that the interaction between Plk1 and Hbo1 is mitosis-specific and that Plk1 phosphorylates Hbo1 on Ser-57 in vitro and in vivo. During mitosis, Cdk1 phosphorylates Hbo1 on Thr-85/88, creating a docking site for Plk1 to be recruited. Significantly, the overexpression of Hbo1 mutated at the Plk1 phosphorylation site (S57A) leads to cell-cycle arrest in the G1/S phase, inhibition of chromatin loading of the minichromosome maintenance (Mcm) complex, and a reduced DNA replication rate. Similarly, Hbo1 depletion results in decreased DNA replication and a failure of Mcm complex binding to chromatin, both of which can be partially rescued by the ectopic expression of WT Hbo1 but not Hbo1-S57A. These results suggest that Plk1 phosphorylation of Hbo1 may be required for prereplicative complex (pre-RC) formation and DNA replication licensing.

SUBMITTER: Wu ZQ

PROVIDER: S-EPMC2538859 | biostudies-other | 2008 Feb

REPOSITORIES: biostudies-other

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Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing.

Wu Zhao-Qiu ZQ Liu Xiaoqi X

Proceedings of the National Academy of Sciences of the United States of America 20080204 6

In a search for Polo-like kinase 1 (Plk1)-interacting proteins using a yeast two-hybrid system, we have identified histone acetyltransferase binding to the origin recognition complex 1 (Hbo1) as a potential Plk1 target. Here, we show that the interaction between Plk1 and Hbo1 is mitosis-specific and that Plk1 phosphorylates Hbo1 on Ser-57 in vitro and in vivo. During mitosis, Cdk1 phosphorylates Hbo1 on Thr-85/88, creating a docking site for Plk1 to be recruited. Significantly, the overexpressio ...[more]

PMID: 18250300

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Project description:Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation.Significance statementEach time a eukaryotic cell divides (by mitosis) it must duplicate its chromosomal DNA exactly once to ensure that one full copy is passed to each resulting cell. Both under-replication or over-replication result in genome instability and disease or cell death. A key mechanism to prevent over-replication is the temporal separation of loading of the replicative DNA helicase at origins of replication and activation of these same helicases during the cell division cycle. Here we define the mechanism by which phosphorylation of the primary DNA binding protein involved in these events inhibits helicase loading. Our studies identify multiple steps of inhibition and provide new insights into the mechanism of helicase loading in the uninhibited condition.

Dataset Information

Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing.

Publications

Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing.

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