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Updated: Feb 3, 2026
Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
Published on: November 5, 2012
The Yeast DNA Damage Checkpoint Kinase Rad53 Targets the Exoribonuclease, Xrn1
Jessica P Lao1, Katie M Ulrich1, Jeffrey R Johnson2
1Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158.
Abstract:
The highly conserved DNA damage response (DDR) pathway monitors the genomic integrity of the cell and protects against genotoxic stresses. The apical kinases, Mec1 and Tel1 (ATR and ATM in human, respectively), initiate the DNA damage signaling cascade through the effector kinases, Rad53 and Chk1, to regulate a variety of cellular processes including cell cycle progression, DNA damage repair, chromatin remodeling, and transcription. The DDR also regulates other cellular pathways, but direct substrates and mechanisms are still lacking. Using a mass spectrometry-based phosphoproteomic screen in Saccharomyces cerevisiae, we identified novel targets of Rad53, many of which are proteins that are involved in RNA metabolism. Of the 33 novel substrates identified, we verified that 12 are directly phosphorylated by Rad53 in vitro: Xrn1, Gcd11, Rps7b, Ded1, Cho2, Pus1, Hst1, Srv2, Set3, Snu23, Alb1, and Scp160. We further characterized Xrn1, a highly conserved 5' exoribonuclease that functions in RNA degradation and the most enriched in our phosphoproteomics screen. Phosphorylation of Xrn1 by Rad53 does not appear to affect Xrn1's intrinsic nuclease activity in vitro, but may affect its activity or specificity in vivo.
Insights
The DNA damage response (DDR) pathway protects genomic integrity. Researchers identified novel Rad53 targets involved in RNA metabolism, revealing new DDR regulatory mechanisms.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The DNA damage response (DDR) pathway is crucial for maintaining genomic stability against various stresses.
- Key apical kinases (Mec1/ATR, Tel1/ATM) and effector kinases (Rad53, Chk1) orchestrate cellular responses.
- While DDR regulates diverse processes, many direct substrates and mechanisms remain uncharacterized.
Purpose of the Study:
- To identify novel direct substrates of the effector kinase Rad53 within the DDR pathway.
- To investigate the role of Rad53 in regulating RNA metabolism through its newly identified targets.
- To characterize the functional impact of Rad53-mediated phosphorylation on specific substrates, such as Xrn1.
Main Methods:
- Utilized mass spectrometry-based phosphoproteomics in *Saccharomyces cerevisiae* to screen for Rad53 targets.
- Performed *in vitro* phosphorylation assays to validate direct Rad53 substrates.
- Characterized the enzymatic activity and potential regulatory effects of Rad53 phosphorylation on the exoribonuclease Xrn1.
Main Results:
- Identified 33 novel Rad53 substrates, with a significant proportion involved in RNA metabolism.
- Validated 12 direct Rad53 phosphorylation targets *in vitro*, including Xrn1, Gcd11, and Ded1.
- Found that Rad53 phosphorylation of Xrn1 does not alter its intrinsic nuclease activity *in vitro* but may impact its *in vivo* function.
Conclusions:
- Rad53 directly phosphorylates numerous proteins involved in RNA metabolism, expanding the known regulatory scope of the DDR.
- The study provides a comprehensive list of novel Rad53 targets, offering new avenues for DDR research.
- Phosphorylation of Xrn1 by Rad53 suggests a regulatory mechanism that influences RNA processing or degradation pathways within the cell.
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