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A role for the phosphorylation of hRad9 in checkpoint signaling
Robert P St Onge1, Blair D A Besley, Jennifer L Pelley
1Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, Ontario, Canada.
Insights
Human genome integrity relies on cell cycle checkpoints. This study reveals hRad9 phosphorylation is critical for DNA damage response and cell cycle regulation, impacting interactions with TopBP1.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Cell cycle checkpoints are crucial for maintaining human genome integrity by halting cell cycle progression upon DNA damage.
- The hRad9-hRad1-hHus1 complex is implicated in detecting DNA structural abnormalities.
- hRad9 undergoes significant phosphorylation, influenced by DNA damage and cell cycle stage.
Purpose of the Study:
- To investigate the specific phosphorylation sites of hRad9 and their regulation by Cdc2.
- To identify novel phosphorylation sites on hRad9 and their role in DNA damage response.
- To elucidate the functional significance of hRad9 phosphorylation in checkpoint signaling and DNA damage response.
Main Methods:
- Phosphorylation site analysis of hRad9, including identification of Cdc2-dependent sites.
- Creation and characterization of nonphosphorylatable hRad9 mutants.
- Investigation of hRad9 mutant interactions with TopBP1 and assessment of cellular response to DNA damage.
Main Results:
- Thr292 of hRad9 is phosphorylated by Cdc2 during mitosis, with other sites (Ser277, Ser328, Ser336, Thr355) also potentially regulated by Cdc2.
- Ser387 was identified as a constitutive phosphorylation site and is essential for DNA damage-induced hyperphosphorylation.
- Nonphosphorylatable hRad9 mutants disrupted the hRad9-TopBP1 interaction and impaired DNA damage response during S phase.
Conclusions:
- hRad9 phosphorylation is vital for effective checkpoint signaling and cellular response to DNA damage.
- Specific phosphorylation events on hRad9, particularly at Ser387, are critical for its function in DNA damage response pathways.
- Dysregulation of hRad9 phosphorylation, as seen with nonphosphorylatable mutants, compromises genome stability by affecting interactions with key proteins like TopBP1.
Abstract:
The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase.