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The replication factory targeting sequence/PCNA-binding site is required in G(1) to control the phosphorylation
1Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy.
Abstract:
The recruitment of DNA ligase I to replication foci in S phase depends on a replication factory targeting sequence that also mediates the interaction with proliferating cell nuclear antigen (PCNA) in vitro. By exploiting a monoclonal antibody directed at a phospho-epitope, we demonstrate that Ser66 of DNA ligase I, which is part of a strong CKII consensus site, is phosphorylated in a cell cycle-dependent manner. After dephosphorylation in early G(1), the level of Ser66 phosphorylation is minimal in G(1), increases progressively in S and peaks in G(2)/M phase. The analysis of epitope-tagged DNA ligase I mutants demonstrates that dephosphorylation of Ser66 requires both the nuclear localization and the PCNA-binding site of the enzyme. Finally, we show that DNA ligase I and PCNA interact in vivo in G(1) and S phase but not in G(2)/M. We propose that dephosphorylation of Ser66 is part of a novel control mechanism to establish the pre-replicative form of DNA ligase I.
Insights
DNA ligase I phosphorylation at Ser66 is cell cycle-dependent, peaking in G2/M. Dephosphorylation requires nuclear localization and PCNA binding, regulating enzyme activity for DNA replication.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Replication
Background:
- DNA ligase I is crucial for DNA replication and repair.
- Its recruitment to replication foci involves interaction with proliferating cell nuclear antigen (PCNA).
- The regulation of DNA ligase I activity during the cell cycle is not fully understood.
Purpose of the Study:
- To investigate the cell cycle-dependent regulation of DNA ligase I phosphorylation.
- To identify the specific site of phosphorylation and its functional significance.
- To elucidate the relationship between DNA ligase I, PCNA, and cell cycle progression.
Main Methods:
- Utilized a phospho-epitope-specific monoclonal antibody to detect Ser66 phosphorylation.
- Analyzed epitope-tagged DNA ligase I mutants to study dephosphorylation requirements.
- Investigated in vivo interactions between DNA ligase I and PCNA using various cell cycle phases.
Main Results:
- Demonstrated cell cycle-dependent phosphorylation of DNA ligase I at Ser66, a CKII consensus site.
- Showed that Ser66 dephosphorylation requires nuclear localization and the PCNA-binding site.
- Confirmed in vivo interaction between DNA ligase I and PCNA during G1 and S phases, but not in G2/M.
Conclusions:
- Propose that Ser66 dephosphorylation is a novel regulatory mechanism for DNA ligase I.
- This mechanism contributes to establishing the pre-replicative form of DNA ligase I.
- Suggests a dynamic interplay between DNA ligase I, PCNA, and cell cycle control.