Telomeres are specialized chromatin structures that protect chromosome ends. Critical among telomere proteins are those that bind the telomeric single-strand DNA (ssDNA) overhangs. These proteins are thought to differ among eukaryotes.

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (γH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, γH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. γH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that γH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to γH2A is crucial in the absence of Rqh1Sgs1, a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund–Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.

Ultra-deep targeted sequencing (UDT-Seq) can identify subclonal somatic mutations in tumor samples. Early assays' limited breadth and depth restrict their clinical utility. Here, we target 71 kb of mutational hotspots in 42 cancer genes. We present novel methods enhancing both laboratory workflow and mutation detection. We evaluate UDT-Seq true sensitivity and specificity (> 94% and > 99%, respectively) for low prevalence mutations in a mixing experiment and demonstrate its utility using six tumor samples. With an improved performance when run on the Illumina Miseq, the UDT-Seq assay is well suited for clinical applications to guide therapy and study clonal selection in heterogeneous samples.

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...