SummaryThe analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C-terminus. Tagged

dRibonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1 ؉ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ϳ30-fold and decreases dTTP ϳ4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1⌬ mutant, fission yeast dcd1⌬ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1⌬ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the ␥H2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1⌬ cells. We propose that replication forks stall and collapse in dcd1⌬ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.T he accurate duplication of a eukaryotic genome demands abundant supplies of deoxyribonucleoside triphosphates (dNTPs), which are the building blocks of DNA. Much of the burden for providing both ample and balanced pools of dNTPs falls to two allosteric enzymes: ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) (22,31,40). RNR plays essential roles in the de novo biosynthesis of all four dNTPs required for DNA synthesis, while dCMP deaminase is specifically involved in the production of dTTP (Fig. 1). Whereas the physiological consequences of RNR defects have been investigated in great detail (9,15,47), the effects of dCMP deaminase deficiency are much less well understood, despite its presumptive key roles in efficient genome duplication and in influencing the outcomes of nucleoside-based antitumor and antiviral therapies (24,25,34).Investigations of dCMP deaminase in a genetically tractable organism have been carried out with the budding yeast Saccharomyces cerevisiae (29, 38). As predicted, null mutations of the DCD1 dCMP deaminase gene significantly increase dCTP and decrease dTTP pools, resulting in an ϳ125-fold increase in the dCTP/dTTP ratio. Surprisingly, these dNTP pool imbalances do not reduce growth rates or have other obvious effects but rather modestly increase mutagenesis rates (29). In contrast, chemical or mutational inhibition of RNR slows DNA replication and activates checkpoint responses. Thus, RNR and dCMP deaminase deficiencies have quite different effects in Saccharomyces cerevisiae.The fission yeast Schizos...

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3′→5′ proofreading activity and its preferred substrates are small gaps with a 5′-phosphate group. Similarly to Polμ, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1–2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polμ, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polμ. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polβ and Polλ, and unlike Polμ, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polβ, Polλ and Polμ.