ATR-dependent pathways control hEXO1 stability in response to stalled forks

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Nucleases are crucial for DNA synthesis, recombination, and repair.
• Human exonuclease 1 (hEXO1) is known to be phosphorylated upon DNA replication stress, leading to its ubiquitination and proteasomal degradation.

Purpose of the Study:

• To identify the signaling pathway responsible for transducing stalled-replication signals to hEXO1.
• To characterize the phosphorylation sites on hEXO1 and their role in protein regulation.

Main Methods:

• Utilized chemical inhibitors, RNA interference, and ATM- and ATR-deficient cell lines.
• Employed mass spectrometry to identify phosphorylation sites on hEXO1.
• Generated and analyzed single- and multiple-point mutants of hEXO1.
• Developed an antibody against a specific phosphorylated residue (pS714).

Main Results:

• hEXO1 phosphorylation in response to stalled replication is dependent on ATR (Ataxia Telangiectasia and Rad3-related protein).
• Mass spectrometry identified nine basal phosphorylation sites and three additional sites induced by hydroxyurea (HU) treatment.
• Mutating the HU-induced phosphorylation sites partially rescued HU-dependent degradation and stabilized hEXO1.
• An antibody targeting pS714 confirmed its phosphorylation upon HU treatment, but not IR (ionizing radiation).

Conclusions:

• The ATR pathway is essential for transducing stalled-replication signals to hEXO1.
• Specific phosphorylation sites on hEXO1 regulate its degradation and stability.
• The developed antibody against pS714 is a valuable tool for monitoring DNA replication stress signaling.