Spd1 accumulation causes genome instability independently of ribonucleotide reductase activity but functions to protect the genome when deoxynucleotide pools are elevated

Abstract

Cullin4, Ddb1 and Cdt2 are core subunits of the ubiquitin ligase complex CRL4(Cdt2), which controls genome stability by targeting Spd1 for degradation during DNA replication and repair in fission yeast. Spd1 has an inhibitory effect on ribonucleotide reductase (RNR), the activity of which is required for deoxynucleotide (dNTP) synthesis. The failure to degrade Spd1 in mutants where CRL4(Cdt2) is defective leads to DNA integrity checkpoint activation and dependency. This correlates with a lower dNTP pool. Pools are restored in a spd1-deleted background and this also suppresses checkpoint activation and dependency. We hypothesized that fission yeast with RNR hyperactivity would display a mutator phenotype on their own, but also possibly repress aspects of the phenotype associated with the inability to target Spd1 for degradation. Here, we report that a mutation in the R1 subunit of ribonucleotide reductase cdc22 (cdc22-D57N), which alleviated allosteric feedback inhibition, caused a highly elevated dNTP pool that was further increased by deleting spd1. The Δspd1 cdc22-D57N double mutant had elevated mutation rates and was sensitive to damaging agents that cause DNA strand breaks, demonstrating that Spd1 can protect the genome when dNTP pools are high. In ddb1-deleted cells, cdc22-D57N also potently elevated RNR activity, but failed to allow cell growth independently of the intact checkpoint. Our results provide evidence that excess Spd1 interferes with other functions in addition to its inhibitory effect on ribonucleotide reduction to generate replication stress and genome instability.

Keywords: Checkpoint kinase; Ddb1; Genome instability; Ribonucleotide reductase; Spd1.

Figures

Fig. 1.

cdc22-D57N does not suppress checkpoint activation and meiosis in Δddb1. (A) Cdc22 levels increase upon γ-irradiation. Western blots showing the levels of Suc22, Cdc22–GFP and Cdc2 in exponentially growing cells exposed to ionizing radiation (500 Gy) at time point zero. Samples were collected at the indicated time points (minutes). (B) Cds1 checkpoint kinase activation by excess Spd1 is not suppressed by cdc22-D57N. As marker for DNA-structure-dependent checkpoint activation, Cds1 kinase activity was monitored after immunoprecipitation from the indicated protein extracts by determining its ability to phosphorylate myelin basic protein (32P-MBP), as shown in the upper panel. The lower panel is a western blot for precipitated Cds1. (C) cdc22-D57N does not suppress checkpoint dependency of Δddb1cells. Serial dilutions of the indicated strains were spotted onto solid rich medium and incubated for 3 days at the indicated temperatures before photography. (D) dNTPs are limiting for repair upon rad3ts inactivation at 36°C. Experiments were performed as in C, but included treatment with MMS as indicated. (E) Formation of Ssb1–GFP foci in Δddb1 cells was suppressed by Δspd1 but not by cdc22-D57N. The Ssb1–GFP fusion protein was expressed from the nmt41 promoter in exponentially growing cells of indicated genotypes and detected by fluorescent microscopy. Yellow arrows point to distinct foci. The percentage of cells with at least one nuclear focus is indicated below the micrographs. (F) Western blot showing Cdt1 and tubulin content in the indicated strains.

Fig. 2.

Cell survival of strains with high dNTP pools in response to DNA-damaging agents. DNA damage sensitivity was evaluated as the ability of the indicated strains to grow on plates containing DNA-damaging drugs. Serial dilutions were spotted onto YEA containing the indicated amounts of drugs and were incubated for 3 days at 30°C. (A) The Δspd1 cdc22-D57N was hypersensitive to MMS, and (B) to CPT. (C) Both cdc22-D57N and Δspd1 cdc22-D57N were more resistant to 4-NQO than wild type. (D) The Δddb1 Δspd1 cdc22-D57N strain was hypersensitive to MMS.

Fig. 3.

The DNA damage sensitivity of Δspd1 cdc22-D57N strains was not suppressed by inactivation of factors involved in translesion DNA synthesis. Serial dilutions were spotted onto YEA containing the indicated amounts of MMS and were incubated for 3 days at 30°C. (A) Inactivation of rev3 or rev7 and (B) inactivation of kpa1 or rev1 in Δspd1 cdc22-D57N background rendered cells more sensitive to MMS.