The Schizosaccharomyces pombe Replication Inhibitor Spd1 Regulates Ribonucleotide Reductase Activity and dNTPs by Binding to the Large Cdc22 Subunit

Olga Chilkova,Pelle Håkansson,Lars Thelander,Lina Dahl,Vladimir Domkin

doi:10.1074/jbc.m511716200

Abstract

Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.

Highlights

Null mutants of the fission yeast csn1-d and csn2-d signalosome subunits are slow growing and have a prolonged S-phase [3]
Because the phenotype of spd1 overexpression resembled the phenotype of csn1-d or csn2-d mutants, Liu et al combined the deletion of spd1 with either of the signalosome subunit mutants, and in both cases, loss of spd1 restored the normal cell cycle profile. They concluded from these experiments that Spd1p inhibits Ribonucleotide reductase (RNR) activity and that the signalosome complex is required to release the inhibition during DNA replication or repair
To increase the supply of deoxyribonucleotides during DNA replication or repair, Spd1p is destroyed by the signalosome; this leads to a delocalization of the Suc22p from the nucleus to the cytoplasm where it combines with the Cdc22p to form active RNR

Summary

Introduction

Null mutants of the fission yeast csn1-d and csn2-d signalosome subunits are slow growing and have a prolonged S-phase [3]. To increase the supply of deoxyribonucleotides during DNA replication or repair, Spd1p is destroyed by the signalosome; this leads to a delocalization of the Suc22p from the nucleus to the cytoplasm where it combines with the Cdc22p to form active RNR.

Results

Conclusion