The ribonucleotide reductase inhibitor, Sml1, is sequentially phosphorylated, ubiquitylated and degraded in response...

Area of Science:

• Molecular Biology
• Cellular Biology
• Genetics

Background:

• Ribonucleotide reductase (RNR) activity is tightly regulated to maintain genome integrity during genotoxic stress.
• The Mec1/Rad53/Dun1 kinase cascade controls RNR, including the RNR inhibitor Sml1.
• DNA damage induces Sml1 degradation, increasing dNTP pools via RNR.

Purpose of the Study:

• To investigate the molecular requirements for Sml1 degradation following DNA damage.
• To identify specific phosphorylation sites on Sml1 critical for its in vivo regulation.
• To elucidate the ubiquitylation and proteasomal pathways involved in Sml1 turnover.

Main Methods:

• Site-directed mutagenesis to identify critical phosphorylation sites on Sml1.
• Analysis of Sml1 degradation in response to DNA damage using mutant strains.
• Investigating the role of the 26S proteasome, E2/E3 ligases (Rad6, Ubr2), and Mub1 in Sml1 degradation.

Main Results:

• Several in vivo phosphorylation sites on Sml1 were identified as essential for its degradation after DNA damage.
• Mutating these sites in Sml1 leads to lethality in conjunction with an rnr1-W688G mutation.
• Sml1 degradation is dependent on the 26S proteasome, the E2 enzyme Rad6, the E3 ligase Ubr2, and Mub1.

Conclusions:

• Sml1 degradation is a critical DNA damage response pathway regulated by specific phosphorylation events.
• The Rad6/Ubr2/Mub1 complex plays a key role in the ubiquitylation and proteasomal degradation of phosphorylated Sml1.
• Understanding Sml1 regulation is vital for maintaining cell survival and genome stability under genotoxic stress.