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Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling
Soo-Jung Lee1, Marc F Schwartz, Jimmy K Duong
1Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510,USA.
Abstract:
Budding yeast Rad53 is an essential protein kinase that is phosphorylated and activated in a MEC1- and TEL1-dependent manner in response to DNA damage. We studied the role of Rad53 phosphorylation through mutation of consensus phosphorylation sites for upstream kinases Mec1 and Tel1. Alanine substitution of the Rad53 amino-terminal TQ cluster region reduced viability and impaired checkpoint functions. These substitution mutations spared the basal interaction with Asf1 and the DNA damage-induced interactions with Rad9. However, they caused a decrease in DNA damage-induced Rad53 kinase activity and an impaired interaction with the protein kinase Dun1. The Dun1 FHA (Forkhead-associated) domain recognized the amino-terminal TQ cluster of Rad53 after DNA damage or replication blockade. Thus, the phosphorylation of Rad53 by upstream kinases is important not only for Rad53 activation but also for creation of an interface between Rad53 and Dun1.
Insights
DNA damage activates Rad53 protein kinase in budding yeast. Phosphorylation of Rad53 by Mec1 and Tel1 is crucial for its activation and interaction with Dun1, ensuring proper DNA repair and checkpoint functions.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Rad53 is an essential protein kinase in budding yeast.
- Rad53 activation and phosphorylation are dependent on MEC1 and TEL1 in response to DNA damage.
Purpose of the Study:
- To investigate the role of Rad53 phosphorylation by Mec1 and Tel1.
- To understand how phosphorylation affects Rad53 interactions and kinase activity.
Main Methods:
- Site-directed mutagenesis of Rad53 phosphorylation sites (TQ cluster).
- Assessing budding yeast viability and checkpoint functions.
- Analyzing protein-protein interactions (Rad53-Asf1, Rad53-Rad9, Rad53-Dun1) using biochemical assays.
- Measuring Rad53 kinase activity post-DNA damage.
Main Results:
- Mutating Rad53's amino-terminal TQ cluster reduced viability and impaired checkpoint functions.
- These mutations did not affect basal Asf1 interaction or DNA damage-induced Rad9 interaction.
- Mutations decreased DNA damage-induced Rad53 kinase activity and impaired Dun1 interaction.
- The Dun1 Forkhead-associated (FHA) domain recognizes the phosphorylated Rad53 TQ cluster.
Conclusions:
- Rad53 phosphorylation by Mec1 and Tel1 is essential for its activation.
- Phosphorylation creates a binding interface for Dun1, facilitating checkpoint signaling.
- This phosphorylation is critical for efficient DNA damage response and cell viability.