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

Andreson BL, Gupta A, Georgieva BP, Rothstein R - Nucleic Acids Res. (2010)

Bottom Line: Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress.Degradation of Sml1 is dependent on the 26S proteasome.We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress. The Mec1/Rad53/Dun1 DNA damage response kinase cascade exhibits multifaceted controls over RNR activity including the regulation of the RNR inhibitor, Sml1. After DNA damage, Sml1 is degraded leading to the up-regulation of dNTP pools by RNR. Here, we probe the requirements for Sml1 degradation and identify several sites required for in vivo phosphorylation and degradation of Sml1 in response to DNA damage. Further, in a strain containing a mutation in Rnr1, rnr1-W688G, mutation of these sites in Sml1 causes lethality. Degradation of Sml1 is dependent on the 26S proteasome. We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4.

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A model for Sml1 regulation in response to DNA damage. Prior to modification, Sml1 is bound to Rnr1 and inhibits RNR activity. Following DNA damage, Dun1 is activated and phosphorylates Sml1 on serines 56, 58, 60 and/or 61. Phosphorylation promotes ubiquitylation of Sml1 by the Rad6–Ubr2–Mub1 complex, targeting Sml1 for degradation by the 26S proteasome. The released Rnr1 associates with Rnr2 and Rnr4 to form an active RNR enzyme allowing the production of dNTPs.
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Figure 6: A model for Sml1 regulation in response to DNA damage. Prior to modification, Sml1 is bound to Rnr1 and inhibits RNR activity. Following DNA damage, Dun1 is activated and phosphorylates Sml1 on serines 56, 58, 60 and/or 61. Phosphorylation promotes ubiquitylation of Sml1 by the Rad6–Ubr2–Mub1 complex, targeting Sml1 for degradation by the 26S proteasome. The released Rnr1 associates with Rnr2 and Rnr4 to form an active RNR enzyme allowing the production of dNTPs.

Mentions: Taken together, our results can be summarized in a model describing Sml1 modifications that target the protein for degradation in response to DNA damage (Figure 6). In this model, before DNA damage, Sml1 is bound to Rnr1, inhibiting RNR activity. Following DNA damage, the Mec1/Rad53/Dun1 kinase cascade is activated and phosphorylates Sml1. This phosphorylation triggers a conformational change in Sml1 leading to its dissociation from Rnr1. Phosphorylated Sml1 is recognized by the Rad6–Ubr2–Mub1 E2/E3 ligase complex, which ubiquitylates Sml1 targeting it for degradation by the 26S proteasome. In the end, loss of Rnr1 inhibition after Sml1 degradation allows the formation of an active RNR enzyme leading to an increase in the production of dNTPs to facilitate DNA damage repair.Figure 6.


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

Andreson BL, Gupta A, Georgieva BP, Rothstein R - Nucleic Acids Res. (2010)

A model for Sml1 regulation in response to DNA damage. Prior to modification, Sml1 is bound to Rnr1 and inhibits RNR activity. Following DNA damage, Dun1 is activated and phosphorylates Sml1 on serines 56, 58, 60 and/or 61. Phosphorylation promotes ubiquitylation of Sml1 by the Rad6–Ubr2–Mub1 complex, targeting Sml1 for degradation by the 26S proteasome. The released Rnr1 associates with Rnr2 and Rnr4 to form an active RNR enzyme allowing the production of dNTPs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2965251&req=5

Figure 6: A model for Sml1 regulation in response to DNA damage. Prior to modification, Sml1 is bound to Rnr1 and inhibits RNR activity. Following DNA damage, Dun1 is activated and phosphorylates Sml1 on serines 56, 58, 60 and/or 61. Phosphorylation promotes ubiquitylation of Sml1 by the Rad6–Ubr2–Mub1 complex, targeting Sml1 for degradation by the 26S proteasome. The released Rnr1 associates with Rnr2 and Rnr4 to form an active RNR enzyme allowing the production of dNTPs.
Mentions: Taken together, our results can be summarized in a model describing Sml1 modifications that target the protein for degradation in response to DNA damage (Figure 6). In this model, before DNA damage, Sml1 is bound to Rnr1, inhibiting RNR activity. Following DNA damage, the Mec1/Rad53/Dun1 kinase cascade is activated and phosphorylates Sml1. This phosphorylation triggers a conformational change in Sml1 leading to its dissociation from Rnr1. Phosphorylated Sml1 is recognized by the Rad6–Ubr2–Mub1 E2/E3 ligase complex, which ubiquitylates Sml1 targeting it for degradation by the 26S proteasome. In the end, loss of Rnr1 inhibition after Sml1 degradation allows the formation of an active RNR enzyme leading to an increase in the production of dNTPs to facilitate DNA damage repair.Figure 6.

Bottom Line: Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress.Degradation of Sml1 is dependent on the 26S proteasome.We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress. The Mec1/Rad53/Dun1 DNA damage response kinase cascade exhibits multifaceted controls over RNR activity including the regulation of the RNR inhibitor, Sml1. After DNA damage, Sml1 is degraded leading to the up-regulation of dNTP pools by RNR. Here, we probe the requirements for Sml1 degradation and identify several sites required for in vivo phosphorylation and degradation of Sml1 in response to DNA damage. Further, in a strain containing a mutation in Rnr1, rnr1-W688G, mutation of these sites in Sml1 causes lethality. Degradation of Sml1 is dependent on the 26S proteasome. We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4.

Show MeSH
Related in: MedlinePlus