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

Tanya Bondar,Aleksandr Ponomarev,Pradip Raychaudhuri

doi:10.1074/jbc.m312570200

Tanya Bondar, Aleksandr Ponomarev + Show 1 more

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https://doi.org/10.1074/jbc.m312570200

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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 Deltaddb1 strain are remarkably similar to the Deltacsn1 (or Deltacsn2) 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 Deltaddb1 strain. In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the Deltaddb1 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.

Highlights

The Schizosaccharomyces pombe Ddb1 protein is homologous to the DDB1 subunit of the mammalian damaged DNA-binding protein DDB,1 which contains an additional subunit DDB2 [1]
The periodic proteolysis of Spd1 in early S phase releases Suc22, which associates with Cdc22 in the cytoplasm to reconstitute the active ribonucleotide reductase (RNR) that is required for the biosynthesis of dNTPs [8]
S Phase Proteolysis of Spd1 Requires Ddb1—We showed that the S. pombe strain lacking expression of ddb1 exhibited a defective S phase, including slow replication, which could be explained by the increased Cds1 kinase activity in that strain [6]

Summary

Introduction

The Schizosaccharomyces pombe Ddb protein is homologous to the DDB1 subunit of the mammalian damaged DNA-binding protein DDB, which contains an additional subunit DDB2 [1]. The S. pombe Ddb protein has been shown to be required for normal cell growth, progression through the S phase, and proper chromosome segregation [5, 6] It is not clear whether those functions are conserved in the mammalian DDB1 protein, both mammalian and yeast Ddb have been shown to associate with Cop9/signalosome, a large complex with homology to the 19 S lid complex of the proteasome [7, 8]. It was shown that the checkpoint-dependent proteolysis of Spd involved Csn because the S. pombe strains lacking this gene failed to induce proteolysis of Spd after ionizing radiation [8] These observations established a positive role of the S. pombe signalosome in the ubiquitin-proteasomemediated proteolysis of Spd. Our stock Our stock Antony Carr This study Paul Nurse This study mutation of Spd caused a reversal of many of the growthrelated phenotypes of the S. pombe strains lacking expression of csn1 [8, 11]

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