Post-translational modification of the ribonucleotide reductase inhibitor, Sml1, in response to DNA damage.

The production of dNTPs is necessary for DNA replication and genome maintenance in the face of genotoxic stress.

Consequently, dNTP pools fluctuate throughout the cell cycle and increase following DNA damage treatment.

Failure to properly regulate dNTP pools can lead to mutagenesis or cell death.

Therefore, it is crucial that cells maintain appropriate levels of dNTPs throughout the cell cycle and in response to DNA damage.

The concentration of dNTPs is largely controlled by precise regulation of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step in dNTP synthesis.

The regulation of RNR is multifaceted and is largely controlled by the DNA damage checkpoint that includes the Mec1, Rad53 and Dun1 kinases.

In Saccharomyces cerevisiae one level of RNR regulation is through a protein inhibitor Sml1.

Sml1 binds to RNR and inhibits the activity of the enzyme.

During S phase and following DNA damage treatment, Sml1 is phosphorylated by Dun1 and degraded.

The studies described here identify two independent in vivo Dun1 phosphorylation events that are required for timely degradation of Sml1 following DNA damage treatment.

We also show that Sml1 is ubiquitylated and its degradation depends on the 26S proteasome.

Interestingly, YFP-Sml1, which usually localizes to the cytoplasm and is excluded from the nucleus, transiently relocalizes to the nucleus prior to degradation by the proteasome.

Mutations that prevent the first phosphorylation event, sml1-SA1, completely block phosphorylation, nuclear localization and degradation of Sml1 following DNA damage treatment.

On the other hand, blocking Sml1 phosphorylation, either by mutation (sml1-SA1) or by inhibiting the kinase (dun1Delta), does not inhibit Sml1 monoubiquitylation, indicating that Sml1 monoubiquitylation is independent of Dun1 phosphorylation.

We propose a model in which Sml1 is monoubiquitylated and then phosphorylated by Dun1, causing it to dissociate from RNR and move into the nucleus.

Nuclear-localized Sml1 is phosphorylated a second time by Dun1, which leads to its polyubiquitylation and targeting for degradation by the 26S proteasome.

Study of the mechanism of Sml1 degradation, which is activated by the DNA damage checkpoint, provides insight into DNA damage-induced protein degradation.

The complexity of regulation for this 104-amino acid Sml1 protein is surprising and underscores the importance of accurately maintaining appropriate levels of dNTP pools. Because Sml1 degradation is activated by the DNA damage checkpoint, these data provide insight into the mechanism of DNA damage-induced protein degradation.