Ddb1 Is Required for the Proteolysis of the Schizosaccharomyces pombe Replication Inhibitor Spd1 during S Phase and after DNA Damage

Abstract

Recently we showed that the Schizosaccharomyces pombe ddb1 gene plays a role in S phase progression. A mutant S. pombe strain lacking expression of the ddb1 gene exhibited slow replication through both early and late regions causing a slow S phase phenotype. We attributed the phenotypes in the ddb1 strain to an increased activity of the replication checkpoint kinase Cds1. However, the basis for a high basal Cds1 activity in the ddb1 strain was not clear. It was shown that Ddb1 associates with the Cop9/signalosome. Moreover, the phenotypes of the ⌬ddb1 strain are remarkably similar to the ⌬csn1 (or ⌬csn2) strain that lacks expression of the Csn1 (or Csn2) subunit of the Cop9/signalosome. Cop9/signalosome cooperates with Pcu4 to induce proteolysis of Spd1, which inhibits DNA replication by inhibiting ribonucleotide reductase. Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1. Here we show that a S. pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 in S phase and after DNA damage. Moreover, deletion of the spd1 gene attenuates the Cds1 kinase activity in cells lacking the expression of ddb1, suggesting that an accumulation of Spd1 results in the increase of Cds1 activity in the ⌬ddb1 strain. In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the ⌬ddb1 strain. Our results establish an essential role of Ddb1 in the proteolysis of Spd1. In addition, the observation provides evidence for a functional link between Ddb1 and the Cop9/signalosome.

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

“…In cells encountering DNA damage or replication blockage, R2 becomes redistributed from the nucleus to the cytoplasm and colocalized with the constitutively cytoplasmic R1 subunit in order to increase RNR holoenzyme formation (29,50). The key player in controlling S. pombe R2 nuclear localization is Spd1, which anchors R2 in the nucleus and is targeted to degradation by the Cop9/signalosome in response to genotoxic stress (4,29,43). In S. cerevisiae, the WD40 repeat protein Wtm1 acts as a nuclear anchor of R2 (26,51).…”

“…Spd1 was originally identified in a screen for S-phase inhibitors because its overexpression causes G 1 arrest (49). Spd1 is subjected to checkpoint-dependent phosphorylation and proteolysis in response to DNA damage (4,29). Its destruction has been found to correlate with redistribution of R2 from the nucleus to the cytoplasm (29,43).…”

Break-induced ATR and Ddb1–Cul4Cdt2 ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

“…One function of this complex in S. pombe is to ubiquitylate and promote the degradation of Spd1, a small inhibitor protein of RNR (Bondar et al 2004;Holmberg et al 2005). To establish whether loss of Spd1 degradation in ddb1D and cdt2D leads to the observed repair defects, spd1 + was deleted in ddb1D and cdt2D backgrounds, thus bypassing the requirement for a functional Ddb1-Cul4 Cdt2 ubiquitin ligase complex to degrade Spd1.…”

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

“…In Schizosaccharomyces pombe, R2 redistribution appears to involve both nuclear export and degradation of the nuclear anchor protein Spd1, an S-phase inhibitor (13,(49)(50)(51)(52). In this study, we set out to determine the mechanism of DNA damage-induced ␤␤Ј redistribution by identifying proteins that have important roles in ␤␤Ј nuclear localization.…”