Ixr1 Is Required for the Expression of the Ribonucleotide Reductase Rnr1 and Maintenance of dNTP Pools

Olga Tsaponina,Andrei Chabes,Emad Barsoum,Stefan U Åström,Nancy Maizels

doi:10.1371/journal.pgen.1002061

Olga Tsaponina, Andrei Chabes + Show 3 more

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https://doi.org/10.1371/journal.pgen.1002061

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Journal: PLoS Genetics	Publication Date: May 5, 2011
Citations: 128	License type: CC BY 4.0

Affiliation: Umeå University, Stockholm University

Abstract

The Saccharomyces cerevisiae Dun1 protein kinase is a downstream target of the conserved Mec1-Rad53 checkpoint pathway. Dun1 regulates dNTP pools during an unperturbed cell cycle and after DNA damage by modulating the activity of ribonucleotide reductase (RNR) by multiple mechanisms, including phosphorylation of RNR inhibitors Sml1 and Dif1. Dun1 also activates DNA-damage-inducible genes by inhibiting the Crt1 transcriptional repressor. Among the genes repressed by Crt1 are three out of four RNR genes: RNR2, RNR3, and RNR4. The fourth RNR gene, RNR1, is also DNA damage-inducible, but is not controlled by Crt1. It has been shown that the deletion of DUN1 is synthetic lethal with the deletion of IXR1, encoding an HMG-box-containing DNA binding protein, but the reason for this lethality is not known. Here we demonstrate that the dun1 ixr1 synthetic lethality is caused by an inadequate RNR activity. The deletion of IXR1 results in decreased dNTP levels due to a reduced RNR1 expression. The ixr1 single mutants compensate for the reduced Rnr1 levels by the Mec1-Rad53-Dun1-Crt1–dependent elevation of Rnr3 and Rnr4 levels and downregulation of Sml1 levels, explaining why DUN1 is indispensible in ixr1 mutants. The dun1 ixr1 synthetic lethality is rescued by an artificial elevation of the dNTP pools. We show that Ixr1 is phosphorylated at several residues and that Ser366, a residue important for the interaction of HMG boxes with DNA, is required for Ixr1 phosphorylation. Ixr1 interacts with DNA at multiple loci, including the RNR1 promoter. Ixr1 levels are decreased in Rad53-deficient cells, which are known to have excessive histone levels. A reduction of the histone gene dosage in the rad53 mutant restores Ixr1 levels. Our results demonstrate that Ixr1, but not Dun1, is required for the proper RNR1 expression both during an unperturbed cell cycle and after DNA damage.

Highlights

Cells experiencing DNA damage or replication blocks activate stress response pathways, or checkpoints, that arrest the cell cycle and facilitate DNA repair
Earlier studies found that simultaneous deletion of DUN1 and IXR1 results in lethality, but the reason for this so-called synthetic lethality is not clear
Ixr1 is implicated in DNA repair based on its ability to bind to DNA modified by the anticancer drug cisplatin

Summary

Introduction

Cells experiencing DNA damage or replication blocks activate stress response pathways, or checkpoints, that arrest the cell cycle and facilitate DNA repair. The key checkpoint protein kinases are Mec (homolog of human ATR) and Rad (homolog of CHK2 and functional homolog of CHK1 in human), reviewed in [1,2]. The essential function of the Mec1-Rad53-Dun pathway is to maintain an adequate supply of dNTPs by regulating the activity of ribonucleotide reductase (RNR) during the normal cell cycle [4,5,6]. Full activation of RNR in S. cerevisiae by the Mec1Rad53-Dun checkpoint in response to DNA damage results in a 6- to 8-fold increase in dNTP concentration [7]. Such increases in dNTP concentration during DNA damage correlate with DNA damage tolerance. Four genes encode yeast RNR: RNR1 and RNR3 encode the large subunit [8,9], and RNR2 and RNR4 encode the small subunit [10,11,12,13]

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