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Published on: March 14, 2021
Protein phosphatase 1 dephosphorylates Orc2
Kyung Yong Lee1, June Sung Bae1, Gwang Su Kim1
1Department of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
Abstract:
Phosphorylation of Thr(116) and Thr(226) on Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2-5) from human chromatin and replication origins. The phosphorylated Orc2 becomes dephosphorylated in the late M phase of the cell cycle. Here we show that protein phosphatase 1 (PP1) dephosphorylates Orc2. Dephosphorylation of Orc2 was accompanied by associating the dissociated Orc subunits with chromatin. Inhibitors of PP1 preferentially inhibited the dephosphorylation of Orc2. The overexpression of the α, β and γ PP1 isoforms decreased the amount of phosphorylated Orc2, and the depletion of these isoforms by RNA interference increased the amount of phosphorylated Orc2. These results suggest that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin.
Insights
Protein phosphatase 1 (PP1) dephosphorylates Orc2, a subunit of the origin recognition complex (ORC). This dephosphorylation promotes the binding of ORC to chromatin, which is essential for DNA replication.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The origin recognition complex (ORC) is crucial for initiating DNA replication in eukaryotes.
- Orc2 phosphorylation by cyclin A/CDK2 during S phase causes dissociation of ORC subunits from chromatin.
- Dephosphorylation of Orc2 in late M phase is necessary for re-associating ORC with chromatin.
Purpose of the Study:
- To identify the phosphatase responsible for Orc2 dephosphorylation.
- To elucidate the role of Orc2 dephosphorylation in ORC-chromatin binding and DNA replication.
Main Methods:
- Biochemical assays to study Orc2 phosphorylation and dephosphorylation.
- Inhibition and overexpression studies of protein phosphatase 1 (PP1) isoforms.
- RNA interference to deplete PP1 isoforms.
Main Results:
- Protein phosphatase 1 (PP1) was identified as the enzyme that dephosphorylates Orc2.
- PP1 inhibition blocked Orc2 dephosphorylation and subsequent Orc subunit re-association with chromatin.
- Overexpression of PP1 isoforms reduced phosphorylated Orc2 levels, while depletion increased them.
Conclusions:
- PP1 plays a critical role in Orc2 dephosphorylation, facilitating the re-binding of the origin recognition complex (ORC) to chromatin.
- This PP1-mediated dephosphorylation is a key regulatory step for efficient DNA replication initiation.
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